Antibodies against claudin 18.2 useful in cancer diagnosis

ABSTRACT

The invention relates to antibodies directed against an epitope located within the C-terminal portion of CLDN18.2 which are useful, for example, in diagnosing cancer and/or in determining whether cancer cells express CLDN18.2.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/397,244, which is a U.S. National Stage Application submitted under35 U.S.C. 371 of International Application Serial NumberPCT/EP2013/001331, filed on May 6, 2013, which claims priority toInternational Application Serial Number PCT/EP2012/001991, filed on May9, 2012. The entire teachings of the above-referenced Applications areincorporated herein by reference.

BACKGROUND

Claudins are integral membrane proteins located within the tightjunctions of epithelia and endothelia. Claudins are predicted to havefour transmembrane segments with two extracellular loops, and N- andC-termini located in the cytoplasm. The claudin (CLDN) family oftransmembrane proteins plays a critical role in the maintenance ofepithelial and endothelial tight junctions and might also play a role inthe maintenance of the cytoskeleton and in cell signaling.

The claudin 18 (CLDN18) molecule is an integral transmembrane protein(tetraspanin) having four membrane spanning hydrophobic regions and twoextracellular loops (loop1 embraced by hydrophobic region 1 andhydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4).CLDN18 exists in two different splice variants, which are described inmouse and in human (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). Thesplice variants (Genbank accession number: splice variant 1 (CLDN18.1):NP_057453, NM_016369, and splice variant 2 (CLDN18.2): NM_001002026,NP_001002026) have a molecular weight of approximately 27.9/27.72 kD.The splice variants CLDN18.1 and CLDN18.2 differ in the N-terminalportion which comprises the first transmembrane (TM) region and loop1,whereas the primary protein sequence of the C-terminus is identical; seeFIG. 1.

CLDN18.1 is selectively expressed on cells of normal lung, whereasCLDN18.2 is expressed only on gastric cells. However, CLDN18.2expression in normal stomach is restricted to the differentiatedshort-lived cells of stomach epithelium. CLDN18.2 expression has beenidentified in various tumor tissues. For example, CLDN18.2 has beenfound to be expressed in pancreatic carcinoma, esophageal carcinoma,gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENTtumors. CLDN18.2 is a valuable target for the prevention and/ortreatment of primary tumors, such as gastric cancer, esophageal cancer,pancreatic cancer, lung cancer such as non small cell lung cancer(NSCLC), ovarian cancer, colon cancer, hepatic cancer, head-neck cancer,and cancers of the gallbladder, and metastases thereof, in particulargastric cancer metastasis such as Krukenberg tumors, peritonealmetastasis, and lymph node metastasis.

The differential expression of CLDN18.2 between cancer and normal cells,its membrane localization, its absence from the vast majority oftoxicity relevant normal tissues, its restriction of expression to adispensable cell population in stomach, differentiated gastric cells,which can be replenished by target-negative stem cells of the stomach,makes CLDN18.2 an attractive target for cancer immunotherapy and the useof antibody-based therapeutics for targeting CLDN18.2 in cancer therapypromises a high level of therapeutic specificity.

The clinical application of CLDN18.2-targeting antibodies faces theobstacle that human CLDN18.2 is highly homologous to human CLDN18.1.CLDN18.2-specific antibodies targeting the N-terminal extracellulardomain of CLDN18.2 displaying sequence differences between humanCLDN18.2 and human CLDN18.1 could successfully be established. Attemptsto produce antibodies targeting the N-terminal portion of CLDN18.2 andhaving properties making them clinically applicable for diagnosticpurposes, e.g. for detection of CLDN18.2 expression in cells of cancertissue sections, failed.

Surprisingly, the present inventors found that antibodies directedagainst a certain epitope located within the C-terminal portion ofCLDN18.2 fulfill the criteria for the diagnostic applicability ofantibodies, in particular for detecting and identifying cells expressingCLDN18.2. Most surprisingly, these antibodies although directed againsta sequence which is identical between CLDN18.1 and CLDN18.2 do nottarget non-cancerous lung cells.

The antibodies of the invention are useful, for example, in diagnosingcancer and/or in determining whether cancer cells express CLDN18.2.Preferably, a cancer disease or a cancer cell is characterized bysurface expression of CLDN18.2. Cancer cells expressing CLDN18.2 aresuitable targets for therapies targeting CLDN18.2 such as therapy withantibodies directed against CLDN18.2. In one embodiment, cancer cellsexpress or aberrantly express CLDN18.2 while the corresponding normalcells do not express CLDN18.2 or express CLDN18.2 at a lower level. Thecells expressing CLDN18.2 are preferably selected from the groupconsisting of tumorigenic gastric, esophageal, pancreatic, lung,ovarian, colon, hepatic, head-neck, and gallbladder cancer cells.

SUMMARY OF THE INVENTION

The present invention relates to an antibody or antigen-binding fragmentthereof which

(i) binds to a peptide having the amino acid sequence TEDEVQSYPSKHDYV(SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6) and/or

(ii) binds to claudin 18.2 (CLDN18.2), wherein said antibody orantigen-binding fragment thereof binds to CLDN18.2 by binding at leastto an epitope within CLDN18.2 having the amino acid sequenceTEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6).

In one embodiment, said CLDN18.2 is cell surface membrane-boundCLDN18.2. In one embodiment, said CLDN18.2 is present on cancer cells,wherein said cancer cells are preferably CLDN18.2 expressing cancercells. In one embodiment, said cancer cells are selected from the groupconsisting of gastric, esophageal, pancreatic, lung, ovarian, colon,hepatic, head-neck, and gallbladder cancer cells. In one embodiment, anantibody or antigen-binding fragment of the invention does not bind tonon-cancerous cells except stomach epithelial cells. In one embodiment,an antibody or antigen-binding fragment of the invention does not bindto non-cancerous lung cells. In one embodiment, an antibody of theinvention is a chimeric, human or humanized antibody. In one embodiment,an antibody of the invention is a monoclonal antibody.

In these and further aspects the present invention relates to anantibody comprising:

(I) an antibody heavy chain comprising:

(i) an antibody heavy chain sequence according to SEQ ID NO: 7 or avariant thereof,

(ii) at least one, preferably two, more preferably all three of the CDRsequences of an antibody heavy chain sequence according to SEQ ID NO: 7or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 10 or a variant thereofand preferably further comprising a CDR1 sequence according to SEQ IDNO: 8 or a variant thereof and/or a CDR2 sequence according to SEQ IDNO: 9 or a variant thereof, and/or

(II) an antibody light chain comprising:

(i) an antibody light chain sequence according to SEQ ID NO: 11 or avariant thereof,

(ii) at least one, preferably two, more preferably all three of the CDRsequences of an antibody light chain sequence according to SEQ ID NO: 11or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 14 or a variant thereofand preferably further comprising a CDR1 sequence according to SEQ IDNO: 12 or a variant thereof and/or a CDR2 sequence according to SEQ IDNO: 13 or a variant thereof.

In the above and further aspects the present invention also relates toan antibody comprising:

(I) an antibody heavy chain comprising:

(i) an antibody heavy chain sequence according to SEQ ID NO: 15 or avariant thereof,

(ii) at least one, preferably two, more preferably all three of the CDRsequences of an antibody heavy chain sequence according to SEQ ID NO: 15or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 18 or a variant thereofand preferably further comprising a CDR1 sequence according to SEQ IDNO: 16 or a variant thereof and/or a CDR2 sequence according to SEQ IDNO: 17 or a variant thereof, and/or

(II) an antibody light chain comprising:

(i) an antibody light chain sequence according to SEQ ID NO: 19 or avariant thereof,

(ii) at least one, preferably two, more preferably all three of the CDRsequences of an antibody light chain sequence according to SEQ ID NO: 19or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 22 or a variant thereofand preferably further comprising a CDR1 sequence according to SEQ IDNO: 20 or a variant thereof and/or a CDR2 sequence according to SEQ IDNO: 21 or a variant thereof.

In preferred embodiments, an antibody of the invention comprises anantibody heavy chain comprising a gamma-1 heavy chain constant region,preferably a human gamma-1 heavy chain constant region and/or comprisesan antibody light chain comprising a kappa light chain constant region.

In the above and further aspects the present invention relates to anantibody produced by or obtainable from a hybridoma cell deposited atthe DSMZ (Inhoffenstr. 7B, 38124 Braunschweig, Germany) and having oneof the following designations and accession numbers:

1. muAB 43-14A, accesssion no. DSM ACC3144, deposited on Oct. 6, 2011;or

2. muAB 35-22A, accesssion no. DSM ACC3143, deposited on Oct. 6, 2011.

Antibodies of the invention are designated herein by referring to thedesignation of the antibody and/or by referring to the clone producingthe antibody, e.g. muAB 43-14A.

Further preferred antibodies are those having the specificity of theantibodies produced by and obtainable from the above-describedhybridomas and, in particular, those comprising an antigen bindingportion or antigen binding site, in particular a variable region,identical or highly homologous to that of the antibodies produced by andobtainable from the above-described hybridomas. It is contemplated thatpreferred antibodies are those having CDR regions either identical orhighly homologous to the CDR regions of antibodies produced by andobtainable from the above-described hybridomas. By “highly homologous”it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1to 3 or 1 or 2 substitutions may be made in each CDR region.Particularly preferred antibodies are the chimerized and humanized formsof the antibodies produced by and obtainable from the above-describedhybridomas.

Thus, an antibody of the invention may be selected from the groupconsisting of (i) an antibody produced by or obtainable from a clonedeposited under the accession no. DSM ACC3144 (muAB 43-14A) or DSMACC3143 (muAB 35-22A), (ii) an antibody which is a chimerized orhumanized form of the antibody under (i), (iii) an antibody which hasthe specificity of the antibody under (i), and (iv) an antibodycomprising the antigen binding portion or antigen binding site of theantibody under (i). The antigen binding portion or antigen binding siteof the antibody under (i) may comprise the variable region of theantibody under (i). Furthermore encompassed by the present invention areantigen-binding fragments of the antibodies described herein.

An antibody of the invention is preferably able to bind to CLDN18.2 inits native, i.e. naturally occurring or non-denatured state, or in itsdenatured state.

In one embodiment, an antibody of the invention is obtainable by amethod comprising the step of immunizing an animal with a peptidecomprising, preferably consisting of the amino acid sequence of SEQ IDNO: 5 or SEQ ID NO: 6, or an immunologically equivalent peptide, or anucleic acid or host cell expressing said peptide. Preferably saidpeptide comprises not more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or20 contiguous amino acids of CLDN18.2.

In one embodiment, an antibody of the invention is obtainable by amethod comprising the step of immunizing an animal with a peptidecomprising, preferably consisting of the amino acid sequence of SEQ IDNO: 24 or SEQ ID NO: 25, or an immunologically equivalent peptide, or anucleic acid or host cell expressing said peptide. Preferably saidpeptide comprises not more than 110, 100, 90, 80, or 75 contiguous aminoacids of CLDN18.2.

Antibodies or antigen-binding fragments of the invention may be coupled,i.e. covalently or non-covalently linked, to other moieties such asdetectable labels.

The present invention also relates to a cell such as a hybridoma cellproducing an antibody as described herein.

Preferred hybridoma cells are those deposited at the DSMZ (Inhoffenstr.7B, 38124 Braunschweig, Germany) and having one of the followingdesignations and accession numbers:

1. muAB 43-14A, accesssion no. DSM ACC3144, deposited on Oct. 6, 2011;or

2. muAB 35-22A, accesssion no. DSM ACC3143, deposited on Oct. 6, 2011.

The present invention also relates to a peptide comprising, preferablyconsisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6,or an immunologically equivalent peptide. Preferably said peptidecomprises not more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20contiguous amino acids of CLDN18.2.

The present invention also relates to a peptide comprising, preferablyconsisting of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25,or an immunologically equivalent peptide, or a nucleic acid or host cellexpressing said peptide. Preferably said peptide comprises not more than110, 100, 90, 80, or 75 contiguous amino acids of CLDN18.2.

The present invention also relates to nucleic acids comprising genes ornucleic acid sequences encoding antibodies or parts thereof, e.g. anantibody chain, or antigen-binding fragments, or peptides as describedherein. Preferably, the nucleic acid of the invention is operativelyattached to expression control elements allowing expression ineukaryotic or prokaryotic cells. Control elements ensuring expression ineukaryotic or prokaryotic cells are well known to those skilled in theart.

The nucleic acids of the invention may be comprised in a vector, e.g., aplasmid, cosmid, virus, bacteriophage or another vector used e.g.conventionally in genetic engineering. The vector may comprise furthergenes such as marker genes which allow for the selection of the vectorin a suitable host cell and under suitable conditions. Furthermore, thevector may comprise expression control elements allowing properexpression of the coding regions in suitable hosts. Such controlelements are known to the artisan and may include a promoter, a splicecassette, and a translation initiation codon.

Methods for construction of nucleic acid molecules, for construction ofvectors comprising nucleic acid molecules, for introduction of vectorsinto appropriately chosen host cells, or for causing or achievingexpression of nucleic acid molecules are well-known in the art.

A further aspect of the present invention relates to a host cellcomprising a nucleic acid or vector as disclosed herein.

A further aspect the present invention relates to the detection ofCLDN18.2 or CLDN18.2-expressing cells or determination of the quantityof CLDN18.2 or CLDN18.2-expressing cells using an antibody orantigen-binding fragment of the invention. CLDN18.2 orCLDN18.2-expressing cells are detected or the quantity of CLDN18.2 orCLDN18.2-expressing cells is determined by detecting or determining theamount of a complex between CLDN18.2 and an antibody or antigen-bindingfragment of the invention.

Formation of a complex indicates the presence of CLDN18.2 orCLDN18.2-expressing cells. Such detection or determination of the amountmay be carried out in a number of ways, including but not limited toimmunodetection using an antibody or antigen-binding fragment of theinvention. Methods for using antibodies to detect peptides or proteinsare well known and include ELISA, competitive binding assays, and thelike. In general, such assays use an antibody or antibody fragment thatspecifically binds the target peptide or protein directly or indirectlybound to a label that provides for detection, e.g. indicator enzymes,radiolabels, fluorophores, or paramagnetic particles. The methods of theinvention allow quantitative and/or qualitative evaluations, e.g.,absolute and/or relative evaluations, of CLDN18.2 levels or of levels ofCLDN18.2-expressing cells.

In one aspect, the present invention relates to a method for detectingCLDN18.2 or determining the quantity of CLDN18.2 in a sample comprisingthe steps of:

(i) contacting a sample with an antibody or antigen-binding fragment ofthe invention or a conjugate of the invention and

(ii) detecting the formation of a complex or determining the quantity ofa complex between the antibody, the antigen-binding fragment or theconjugate and CLDN18.2.

In one embodiment, the sample is a cellular sample, i.e. a samplecomprising cells such as cancer cells. In this embodiment, the complexis preferably formed between the antibody, the antigen-binding fragmentor the conjugate and CLDN18.2 expressed by cells in said sample.

In one aspect, the present invention relates to a method for determiningwhether cells express CLDN18.2 comprising the steps of:

(i) contacting a cellular sample with an antibody or antigen-bindingfragment of the invention or a conjugate of the invention and

(ii) detecting the formation of a complex between the antibody, theantigen-binding fragment or the conjugate and CLDN18.2 expressed bycells in said sample.

In one embodiment, the cells in the sample are cancer cells. The complexis preferably formed between the antibody, the antigen-binding fragmentor the conjugate and CLDN18.2 expressed by cells in said sample.

Further aspects of the present invention relate to methods of diagnosingor classifying diseases by targeting CLDN18.2 using an antibody orantigen-binding fragment of the invention. These methods provide for theselective detection of cells that express CLDN18.2 therebydifferentiating these cells from normal cells not expressing CLDN18.2 ordiseased cells not expressing CLDN18.2. Diseases characterized bydiseased cells expressing CLDN18.2 are treatable by a therapy targetingCLDN18.2 such as therapy with therapeutic antibodies directed againstCLDN18.2. Preferred diseases for a therapy or diagnosis are those inwhich CLDN18.2 is expressed or aberrantly expressed, in particularcancer diseases, such as those described herein.

In one aspect the present invention relates to methods for diagnosis,detection or monitoring, i.e. determining the regression, progression,course and/or onset, of a cancer disease comprising the detection ofCLDN18.2 or CLDN18.2-expressing cells and/or determination of thequantity of CLDN18.2 or CLDN18.2-expressing cells in a biological sampleisolated from a patient using an antibody or antigen-binding fragment ofthe invention. Such methods may be used to detect whether a subject hasa cancer disease or is at (increased) risk of developing a cancerdisease or, for instance, whether a treatment regimen is efficient.

Thus, in one aspect, the present invention relates to a method fordiagnosis, detection or monitoring of cancer comprising the steps of:

(i) contacting a biological sample with an antibody or antigen-bindingfragment of the invention or a conjugate of the invention and

(ii) detecting the formation of a complex and/or determining thequantity of a complex between the antibody, the antigen-binding fragmentor the conjugate and CLDN18.2.

In one embodiment, the biological sample is a cellular sample, i.e. asample comprising cells such as cancer cells. In this embodiment, thecomplex is preferably formed between the antibody, the antigen-bindingfragment or the conjugate and CLDN18.2 expressed by cells in saidsample.

The methods of monitoring according to the invention preferably comprisea detection of and/or determination of the quantity of CLDN18.2 orCLDN18.2-expressing cells in a first sample at a first point in time andin a further sample at a second point in time, wherein the regression,progression, course and/or onset of a tumor disease may be determined bycomparing the two samples.

Typically, the level of CLDN18.2 or level of CLDN18.2-expressing cellsin a biological sample is compared to a reference level, wherein adeviation from said reference level is indicative of the presence and/orstage of a cancer disease in a subject. The reference level may be alevel as determined in a control sample (e.g., from a healthy tissue orsubject, in particular a patient without a cancer disease) or a medianlevel from healthy subjects. A “deviation” from said reference leveldesignates any significant change, such as an increase by at least 10%,20%, or 30%, preferably by at least 40% or 50%, or even more.

Preferably, the presence of CLDN18.2 or CLDN18.2-expressing cells and/ora quantity of CLDN18.2 or CLDN18.2-expressing cells which is increasedcompared to a reference level, e.g. compared to a patient without acancer disease, indicates the presence of or risk for (i.e. a potentialfor a development of) a cancer disease in the patient.

A quantity of CLDN18.2 or CLDN18.2-expressing cells which is decreasedcompared to a biological sample taken earlier from a patient mayindicate a regression, a positive course, e.g. a successful treatment,or a reduced risk for an onset of a cancer disease in a patient.

A quantity of CLDN18.2 or CLDN18.2-expressing cells which is increasedcompared to a biological sample taken earlier from a patient mayindicate a progression, a negative course, e.g. an unsuccessfultreatment, recurrence or metastatic behaviour, an onset or a risk for anonset of a cancer disease in said patient.

In one aspect, the present invention relates to a method for determiningwhether a cancer is treatable by a cancer therapy targeting CLDN18.2comprising the steps of:

(i) contacting a sample comprising cancer cells with an antibody orantigen-binding fragment of the invention or a conjugate of theinvention and

(ii) detecting the formation of a complex between the antibody, theantigen-binding fragment or the conjugate and CLDN18.2.

The complex is preferably formed between the antibody, theantigen-binding fragment or the conjugate and CLDN18.2 expressed bycancer cells in said sample.

Such methods may be used to detect whether a patient is suitable for atherapy involving the targeting of cells expressing CLDN18.2 such as atherapy using antibodies exerting one or more immune effector functionssuch as cytotoxic CLDN18.2 specific antibodies, e.g. antibodies labeledwith a cytotoxic substance such as a toxin or a radiolabel or inducing acell killing mechanism such as CDC or ADCC. Diseases characterized bydiseased cells expressing CLDN18.2 are treatable by a therapy targetingCLDN18.2 such as cancer diseases, in particular those described herein.

In one embodiment of any of the above aspects, the sample, cellularsample or biological sample is from a patient having a cancer disease,being suspected of having or falling ill with a cancer disease or havinga potential for a cancer disease. In one embodiment, the sample,cellular sample or biological sample is from a tissue or organ whereinthe cells when the tissue or organ is free of cancer do notsubstantially express CLDN18.2. Preferably said tissue is a tissue otherthan stomach tissue. Preferably, said tissue is tissue of lung,esophagus, pancreas or breast and the tissue or organ optionally hasalready been diagnosed as being affected by a cancer disease, e.g. byvisual inspection or culture testing of cells of said tissue or organ.In this embodiment, the presence of CLDN18.2 or CLDN18.2-expressingcells and/or a quantity of CLDN18.2 or CLDN18.2-expressing cells whichis increased compared to a reference level, e.g. compared to a patientwithout a tumor disease, may indicate that a patient is suitable for atherapy involving the targeting of cells expressing CLDN18.2.

In one aspect, the invention provides compositions, e.g., diagnosticcompositions, or kits, comprising an antibody or antigen-bindingfragment or a combination of antibodies and/or or antigen-bindingfragments described herein. Such diagnostic compositions or test kitsare useful in the methods of the invention such as the methods fordiagnosis, detection or monitoring of the invention. These kits mayoptionally comprise a detectable label, e.g. indicator enzymes,radiolabels, fluorophores, or paramagnetic particles. Kits may includeinformative pamphlets, for example, pamphlets informing one how to usereagents to practice a method disclosed herein.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sequence alignment of claudin 18 proteins (human/murine)

The sequence alignment shows the high homology between human and mouseclaudin 18.2 and human claudin 18.1 and claudin 18.2.

FIG. 2: Recombinant protein including the C-terminal portion of CLDN18.2(aa191-261) used for immunization of mice

FIG. 3: Sequence-analysis of 43-14A and 35-22A antibodies

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant object”such as a recombinant cell in the context of the present invention isnot occurring naturally.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response is directed and/or is to be generated.Preferably, an antigen in the context of the present invention is amolecule which, optionally after processing, induces an immune reaction,which is preferably specific for the antigen. The term “antigen”includes in particular proteins, peptides, polysaccharides, nucleicacids, especially RNA and DNA, and nucleotides.

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. An epitope of a protein such as a CLDN preferablycomprises a continuous or discontinuous portion of said protein and ispreferably between 5 and 100, preferably between 5 and 50, morepreferably between 8 and 30, most preferably between 10 and 25 aminoacids in length, for example, the epitope may be preferably 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids in length.

The term “discontinuous epitope” as used herein, means a conformationalepitope on a protein antigen which is formed from at least two separateregions in the primary sequence of the protein.

In a preferred embodiment, an antigen is a tumor-associated antigen,such as CLDN18.2, i.e., a constituent of cancer cells which may bederived from the cytoplasm, the cell surface and the cell nucleus, inparticular those antigens which are produced, preferably in largequantity, intracellular or as surface antigens on cancer cells.

In the context of the present invention, the terms “tumor-associatedantigen” or “tumor antigen” relate to proteins that are under normalconditions specifically expressed in a limited number of tissues and/ororgans or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In this context, “alimited number” preferably means not more than 3, more preferably notmore than 2. The tumor-associated antigens in the context of the presentinvention include, for example, differentiation antigens, preferablycell type specific differentiation antigens, i.e., proteins that areunder normal conditions specifically expressed in a certain cell type ata certain differentiation stage, cancer/testis antigens, i.e., proteinsthat are under normal conditions specifically expressed in testis andsometimes in placenta, and germ line specific antigens. In the contextof the present invention, the tumor-associated antigen is preferablyassociated with the cell surface of a cancer cell and is preferably notor only rarely expressed in normal tissues. Preferably, thetumor-associated antigen or the aberrant expression of thetumor-associated antigen identifies cancer cells. In the context of thepresent invention, the tumor-associated antigen that is expressed by acancer cell in a subject, e.g., a patient suffering from a cancerdisease, is preferably a self-protein in said subject. In preferredembodiments, the tumor-associated antigen in the context of the presentinvention is expressed under normal conditions specifically in a tissueor organ that is non-essential, i.e., tissues or organs which whendamaged by the immune system do not lead to death of the subject, or inorgans or structures of the body which are not or only hardly accessibleby the immune system. Preferably, the amino acid sequence of thetumor-associated antigen is identical between the tumor-associatedantigen which is expressed in normal tissues and the tumor-associatedantigen which is expressed in cancer tissues.

Examples for differentiation antigens which ideally fulfill the criteriafor tumor-associated antigens as target structures in tumorimmunotherapy, in particular, in tumor vaccination are the cell surfaceproteins of the claudin family, such as CLDN18.2. Claudins are a familyof proteins that are the most important components of tight junctions,where they establish the paracellular barrier that controls the flow ofmolecules in the intercellular space between cells of an epithelium.Claudins are transmembrane proteins spanning the membrane 4 times withthe N-terminal and the C-terminal end both located in the cytoplasm.

The term “claudin 18” or “CLDN18” preferably relates to human CLDN18 andincludes any splice variants such as CLDN18.1 and CLDN18.2 of CLDN18.CLDN18.1 and CLDN18.2 differ in the N-terminal portion which comprisesthe first transmembrane (TM) region and loop1, whereas the primaryprotein sequence of the C-terminus is identical.

The term “CLDN18.1” preferably relates to human CLDN18.1, and, inparticular, to a protein comprising the amino acid sequence according toSEQ ID NO: 1 of the sequence listing or a variant of said amino acidsequence.

The term “CLDN18.2” preferably relates to human CLDN18.2, and, inparticular, to a protein comprising the amino acid sequence according toSEQ ID NO: 2 of the sequence listing or a variant of said amino acidsequence.

The term “variant” according to the invention refers, in particular, tomutants, splice variants, conformations, isoforms, allelic variants,species variants and species homologs, in particular those which arenaturally present. An allelic variant relates to an alteration in thenormal sequence of a gene, the significance of which is often unclear.Complete gene sequencing often identifies numerous allelic variants fora given gene. A species homolog is a nucleic acid or amino acid sequencewith a different species of origin from that of a given nucleic acid oramino acid sequence.

The terms “CLDN”, “CLDN18”, “CLDN18.1” and “CLDN18.2” shall encompassany posttranslationally modified variants and conformation variants.

CLDN18.2 is selectively expressed in normal tissues in differentiatedepithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancersof various origins, and is particularly suited as target structure forthe development of antibody-mediated cancer immunotherapy due to itsselective expression (no expression in a toxicity relevant normaltissue) and localization to the plasma membrane. For example, CLDN18.2has been found to be expressed in pancreatic carcinoma, esophagealcarcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, andENT tumors. CLDN18.2 is a valuable target for the prevention and/ortreatment of primary tumors, such as gastric cancer, esophageal cancer,pancreatic cancer, lung cancer such as non small cell lung cancer(NSCLC), ovarian cancer, colon cancer, hepatic cancer, head-neck cancer,and cancers of the gallbladder, and metastases thereof, in particulargastric cancer metastasis such as Krukenberg tumors, peritonealmetastasis, and lymph node metastasis. Cells expressing CLDN18.2 arepreferably cancer cells and are, in particular, selected from the groupconsisting of tumorigenic gastric, esophageal, pancreatic, lung,ovarian, colon, hepatic, head-neck, and gallbladder cancer cells.

According to the invention, a cell expressing CLDN18.2 is preferablycharacterized by cell-surface membrane-bound CLDN18.2, i.e. CLDN18.2 isassociated with the cell surface. Furthermore, according to theinvention, cellular CLDN18.2 is preferably cell-surface membrane-boundCLDN18.2. A cell expressing CLDN18.2 or a cell characterized byassociation of CLDN18.2 with its cell surface preferably is a cancercell, preferably a cancer cell from a cancer described herein.

The term “associated with the cell surface” means that atumor-associated antigen such as CLDN18.2 is associated with and locatedat the plasma membrane of a cell, wherein at least a part of thetumor-associated antigen faces the extracellular space of said cell andis accessible from the outside of said cell, e.g., by antibodies locatedoutside the cell. In this context, a part is preferably at least 4,preferably at least 8, preferably at least 12, more preferably at least20 amino acids. The association may be direct or indirect. For example,the association may be by one or more transmembrane domains, one or morelipid anchors, or by the interaction with any other protein, lipid,saccharide, or other structure that can be found on the outer leaflet ofthe plasma membrane of a cell. For example, a tumor-associated antigenassociated with the surface of a cell may be a transmembrane proteinhaving an extracellular portion or may be a protein associated with thesurface of a cell by interacting with another protein that is atransmembrane protein.

“Cell surface” or “surface of a cell” is used in accordance with itsnormal meaning in the art, and thus includes the outside of the cellwhich is accessible to binding by proteins and other molecules.

According to the invention CLDN18.2 is not substantially expressed in acell and is not substantially associated with a cell surface if thelevel of expression and association exceeds the level of expression andassociation in non-cancerous tissue other than stomach by no more than2-fold, preferably 1.5-fold, and preferably does not exceed the level ofexpression and association in said non-cancerous tissue. Preferably,CLDN18.2 is not substantially expressed in a cell and is notsubstantially associated with a cell surface if the level of expressionor association is below the detection limit and/or if the level ofexpression or association is too low to allow binding byCLDN18.2-specific antibodies added to the cells.

According to the invention CLDN18.2 is expressed in a cell and isassociated with a cell surface if the level of expression andassociation exceeds the level of expression and association innon-cancerous tissue other than stomach, preferably by more than 2-fold,preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably,CLDN18.2 is expressed in a cell and is associated with a cell surface ifthe level of expression and association is above the detection limitand/or if the level of expression and association is high enough toallow binding by CLDN18.2-specific antibodies added to the cells.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, and includes any molecule comprising an antigen binding portionthereof. The term “antibody” includes monoclonal antibodies andfragments or derivatives of antibodies, including, without limitation,human antibodies, humanized antibodies, chimeric antibodies, singlechain antibodies, e.g., scFv's and antigen-binding antibody fragmentssuch as Fab and Fab′ fragments and also includes all recombinant formsof antibodies, e.g., antibodies expressed in prokaryotes, unglycosylatedantibodies, and any antigen-binding antibody fragments and derivativesas described herein. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically, the variable region of both light and heavychains mimics the variable regions of antibodies derived from onespecies of mammals, while the constant portions are homologous tosequences of antibodies derived from another. One clear advantage tosuch chimeric forms is that the variable region can conveniently bederived from presently known sources using readily available B-cells orhybridomas from non-human host organisms in combination with constantregions derived from, for example, human cell preparations. While thevariable region has the advantage of ease of preparation and thespecificity is not affected by the source, the constant region beinghuman, is less likely to elicit an immune response from a human subjectwhen the antibodies are injected than would the constant region from anon human source. However the definition is not limited to thisparticular example.

The terms “antigen-binding portion” of an antibody (or simply “bindingportion”) or “antigen-binding fragment” of an antibody (or simply“binding fragment”) refer to one or more fragments of an antibody thatretain the ability to specifically bind to an antigen. It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) Fab fragments, monovalent fragments consisting of the VL,VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fvfragments consisting of the VL and VH domains of a single arm of anantibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546),which consist of a VH domain; (vi) isolated complementarity determiningregions (CDR), and (vii) combinations of two or more isolated CDRs whichmay optionally be joined by a synthetic linker. Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding fragment” of an antibody. A further example isbinding-domain immunoglobulin fusion proteins comprising (i) a bindingdomain polypeptide that is fused to an immunoglobulin hinge regionpolypeptide, (ii) an immunoglobulin heavy chain CH2 constant regionfused to the hinge region, and (iii) an immunoglobulin heavy chain CH3constant region fused to the CH2 constant region. The binding domainpolypeptide can be a heavy chain variable region or a light chainvariable region. The binding-domain immunoglobulin fusion proteins arefurther disclosed in US 2003/0118592 and US 2003/0133939. These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies.

The antibodies described herein may be monoclonal antibodies. The term“monoclonal antibody” as used herein refers to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodydisplays a single binding specificity and affinity. In one embodiment,the monoclonal antibodies are produced by a hybridoma which includes a Bcell obtained from a non-human animal, e.g., mouse, fused to animmortalized cell.

The antibodies described herein may be recombinant antibodies. The term“recombinant antibody”, as used herein, includes all antibodies that areprepared, expressed, created or isolated by recombinant means, such as(a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The invention includes all antibodies and derivatives of antibodies asdescribed herein which for the purposes of the invention are encompassedby the term “antibody”. The term “antibody derivatives” refers to anymodified form of an antibody, e.g., a conjugate of the antibody andanother agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities. Moreover, an isolated antibody may be substantially freeof other cellular material and/or chemicals.

According to the present invention, an antibody is capable of binding toa predetermined target if it has a significant affinity for saidpredetermined target and binds to said predetermined target in standardassays. “Affinity” or “binding affinity” is often measured byequilibrium dissociation constant (K_(D)). Preferably, the term“significant affinity” refers to the binding to a predetermined targetwith a dissociation constant (K_(D)) of 10⁻⁵ M or lower, 10⁻⁶ M orlower, 10⁻⁷ M or lower, 10⁻⁸ M or lower, 10⁻⁹ M or lower, 10⁻¹⁰ M orlower, 10⁻¹¹ M or lower, or 10⁻¹² M or lower.

An antibody is not (substantially) capable of binding to a target if ithas no significant affinity for said target and does not bindsignificantly, in particular does not bind detectably, to said target instandard assays. Preferably, the antibody does not detectably bind tosaid target if present in a concentration of up to 2, preferably 10,more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably,an antibody has no significant affinity for a target if it binds to saidtarget with a K_(D) that is at least 10-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold, or 10⁶-fold higher than the K_(D) for binding to thepredetermined target to which the antibody is capable of binding. Forexample, if the K_(D) for binding of an antibody to the target to whichthe antibody is capable of binding is 10⁻⁷ M, the K_(D) for binding to atarget for which the antibody has no significant affinity would be is atleast 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

An antibody is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not capable of bindingto other targets, i.e. has no significant affinity for other targets anddoes not significantly bind to other targets in standard assays.According to the invention, an antibody is specific for CLDN18.2 if itis capable of binding to CLDN18.2 but is not (substantially) capable ofbinding to other targets, in particular proteins other than claudinproteins, preferably proteins other than CLDN18, in particular proteinsother than CLDN18.2. Preferably, an antibody is specific for CLDN18.2 ifthe affinity for and the binding to such other targets does notsignificantly exceed the affinity for or binding to claudin-unrelatedproteins such as bovine serum albumin (BSA), casein, human serum albumin(HSA) or non-claudin transmembrane proteins such as MHC molecules ortransferrin receptor or any other specified polypeptide. Preferably, anantibody is specific for a predetermined target if it binds to saidtarget with a K_(D) that is at least 10-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold, or 10⁶-fold lower than the K_(D) for binding to atarget for which it is not specific. For example, if the K_(D) forbinding of an antibody to the target for which it is specific is 10⁻⁷ M,the K_(D) for binding to a target for which it is not specific would beat least 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

Binding of an antibody to a target can be determined experimentallyusing any suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N.Y. (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. Antibodiesaccording to the invention include polyclonal and monoclonal antibodiesand include IgG2a (e.g. IgG2a, κ, λ), IgG2b (e.g. IgG2b, κ, λ), IgG3(e.g. IgG3, κ, λ) and IgM antibodies. However, other antibody isotypesare also encompassed by the invention, including IgG1, IgA1, IgA2,secretory IgA, IgD, and IgE antibodies.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

According to the invention, antibodies may be derived from differentspecies, including but not limited to mouse, rat, rabbit, guinea pig andhuman. Antibodies also include chimeric molecules in which an antibodyconstant region derived from one species, preferably human, is combinedwith the antigen binding site derived from another species. Moreover,antibodies include humanized molecules in which the antigen bindingsites of an antibody derived from a non-human species are combined withconstant and framework regions of human origin.

Antibodies can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, e.g., the standard somaticcell hybridization technique of Kohler and Milstein, Nature 256: 495(1975). Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies can beemployed, e.g., viral or oncogenic transformation of B-lymphocytes orphage display techniques using libraries of antibody genes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodiesdirected against CLDN18.2 can be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. These transgenic and transchromosomic miceinclude mice known as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice.” The production ofhuman antibodies in such transgenic mice can be performed as describedin detail for CD20 in WO2004 035607

Yet another strategy for generating monoclonal antibodies is to directlyisolate genes encoding antibodies from lymphocytes producing antibodiesof defined specificity; see e.g. Babcock et al., 1996; A novel strategyfor generating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined specificities. For details ofrecombinant antibody engineering see also Welschof and Kraus,Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies to CLDN18.2, mice can be immunized withcarrier-conjugated peptides derived from the CLDN18.2 sequence, anenriched preparation of recombinantly expressed CLDN18.2 antigen orfragments thereof and/or cells expressing CLDN18.2 or fragments thereof,as described. Alternatively, mice can be immunized with DNA encodingfull length human CLDN18.2 or fragments thereof. In the event thatimmunizations using a purified or enriched preparation of the CLDN18.2antigen do not result in antibodies, mice can also be immunized withcells expressing CLDN18.2, e.g., a cell line, to promote immuneresponses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of anti-CLDN18.2immunoglobulin can be used for fusions. Mice can be boostedintraperitonealy or intravenously with CLDN18.2 expressing cells 3-5days before sacrifice and removal of the spleen to increase the rate ofspecific antibody secreting hybridomas.

To generate hybridomas producing monoclonal antibodies to CLDN18.2,cells from lymph nodes, spleens or bone marrow obtained from immunizedmice can be isolated and fused to an appropriate immortalized cell line,such as a mouse myeloma cell line. The resulting hybridomas can then bescreened for the production of antigen-specific antibodies. Individualwells can then be screened by ELISA for antibody secreting hybridomas.By Immunofluorescence and FACS analysis using CLDN18.2 expressing cells,antibodies with specificity for CLDN18.2 can be identified. The antibodysecreting hybridomas can be replated, screened again, and if stillpositive for anti-CLDN18.2 monoclonal antibodies can be subcloned bylimiting dilution. The stable subclones can then be cultured in vitro togenerate antibody in tissue culture medium for characterization.

Antibodies of the invention can also be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as are well known in the art(Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. Afterintroduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Chimeric antibodies are antibodies, the different portions of which arederived from different animal species, such as those having a variableregion derived from a murine antibody and a human immunoglobulinconstant region. Chimerisation of antibodies is achieved by joining ofthe variable regions of the murine antibody heavy and light chain withthe constant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment, chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment, chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual positions evenly across the variable region. For example,somatic mutations are relatively infrequent in the amino terminalportion of framework region 1 and in the carboxy-terminal portion offramework region 4. Furthermore, many somatic mutations do notsignificantly alter the binding properties of the antibody. For thisreason, it is not necessary to obtain the entire DNA sequence of aparticular antibody in order to recreate an intact recombinant antibodyhaving binding properties similar to those of the original antibody (seeWO 99/45962). Partial heavy and light chain sequences spanning the CDRregions are typically sufficient for this purpose. The partial sequenceis used to determine which germline variable and joining gene segmentscontributed to the recombined antibody variable genes. The germlinesequence is then used to fill in missing portions of the variableregions. Heavy and light chain leader sequences are cleaved duringprotein maturation and do not contribute to the properties of the finalantibody. To add missing sequences, cloned cDNA sequences can becombined with synthetic oligonucleotides by ligation or PCRamplification. Alternatively, the entire variable region can besynthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas may be used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266: 19867-19870); and HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotides approximately at the midpoint of thecorresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assembled into overlapping double stranded setsthat span segments of 150-400 nucleotides. The pools are then used astemplates to produce PCR amplification products of 150-400 nucleotides.Typically, a single variable region oligonucleotide set will be brokendown into two pools which are separately amplified to generate twooverlapping PCR products. These overlapping products are then combinedby PCR amplification to form the complete variable region. It may alsobe desirable to include an overlapping fragment of the heavy or lightchain constant region in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs.

The reconstructed chimerized or humanized heavy and light chain variableregions are then combined with cloned promoter, leader, translationinitiation, constant region, 3′ untranslated, polyadenylation, andtranscription termination sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains. Plasmids for use in construction ofexpression vectors for human IgGκ are described. The plasmids can beconstructed so that PCR amplified V heavy and V kappa light chain cDNAsequences can be used to reconstruct complete heavy and light chainminigenes. These plasmids can be used to express completely human, orchimeric IgG1, Kappa or IgG4, Kappa antibodies. Similar plasmids can beconstructed for expression of other heavy chain isotypes, or forexpression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural features of theanti-CLDN18.2 antibodies described herein, are used to createstructurally related humanized anti-CLDN18.2 antibodies that retain atleast one functional property of the antibodies of the invention, suchas binding to CLDN18.2. More specifically, one or more CDR regions ofmouse monoclonal antibodies can be combined recombinantly with knownhuman framework regions and CDRs to create additional,recombinantly-engineered, humanized anti-CLDN18.2 antibodies.

The ability of an antibody to bind CLDN18.2 can be determined usingstandard binding assays, e.g., ELISA, Western Blot, Immunofluorescenceand Flow cytometric analysis.

ELISA can be used to demonstrate the presence of antibodies in sera ofimmunized mice or binding of monoclonal antibodies to CLDN18.2 proteinor peptides. Peptides or protein used for immunization may be used fordetermining the specificity of hybridoma supernatants or analysing serumtiters.

In order to demonstrate presence of antibodies in sera of immunized miceor binding of monoclonal antibodies to living cells, flow cytometry canbe used. Cell lines expressing naturally or after transfection antigenand negative controls lacking antigen expression (grown under standardgrowth conditions) can be mixed with various concentrations ofmonoclonal antibodies in hybridoma supernatants or in PBS containing 1%FBS, and can be incubated at 4° C. for 30 min. After washing, the APC-or Alexa647-labeled anti IgG antibody can bind to antigen-boundmonoclonal antibody under the same conditions as the primary antibodystaining. The samples can be analyzed by flow cytometry with a FACSinstrument using light and side scatter properties to gate on single,living cells. In order to distinguish antigen-specific monoclonalantibodies from non-specific binders in a single measurement, the methodof co-transfection can be employed. Cells transiently transfected withplasmids encoding antigen and a fluorescent marker can be stained asdescribed above. Transfected cells can be detected in a differentfluorescence channel than antibody-stained cells. As the majority oftransfected cells express both transgenes, antigen-specific monoclonalantibodies bind preferentially to fluorescence marker expressing cells,whereas non-specific antibodies bind in a comparable ratio tonon-transfected cells. An alternative assay using fluorescencemicroscopy may be used in addition to or instead of the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy.

In order to demonstrate presence of anti-CLDN18.2 antibodies in sera ofimmunized mice or binding of monoclonal antibodies to living cellsexpressing CLDN18.2, immunofluorescence microscopy analysis can be used.For example, cell lines expressing either spontaneously or aftertransfection CLDN18.2 and negative controls lacking CLDN18.2 expressionare grown in chamber slides under standard growth conditions in DMEM/F12medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine,100 IU/ml penicillin and 100 μg/ml streptomycin. Cells can then be fixedwith methanol or paraformaldehyde or left untreated. Cells can then bereacted with monoclonal antibodies against CLDN18.2 for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelledanti-mouse IgG secondary antibody (Molecular Probes) under the sameconditions. Cells can then be examined by fluorescence microscopy.

Total CLDN18.2 levels in cells can be observed when cells are methanolfixed or paraformaldehyde fixed and permeabilized with Triton X-100. Inliving cells and non-permeabilized, paraformaldehyde fixed cells surfacelocalization of CLDN18.2 can be examined. Additionally targeting ofCLDN18.2 to tight junctions can be analyzed by co-staining with tightjunction markers such as ZO-1. Furthermore, effects of antibody bindingand CLDN18.2 localization within the cell membrane can be examined.

Anti-CLDN18.2 IgG can be further tested for reactivity with CLDN18.2antigen by Western Blotting. Briefly, cell extracts from cellsexpressing CLDN18.2 and appropriate negative controls can be preparedand subjected to sodium dodecyl sulfate (SDS) polyacrylamide gelelectrophoresis. After electrophoresis, the separated antigens will betransferred to nitrocellulose membranes, blocked, and probed with themonoclonal antibodies to be tested. IgG binding can be detected usinganti-mouse IgG peroxidase and developed with ECL substrate.

Anti-CLDN18.2 mouse IgGs can be further tested for reactivity withCLDN18.2 antigen by Immunohistochemistry in a manner well known to theskilled person, e.g. using paraformaldehyde or acetone fixedcryosections or paraffin embedded tissue sections fixed withparaformaldehyde from non-cancer tissue or cancer tissue samplesobtained from patients during routine surgical procedures or from micecarrying xenografted tumors inoculated with cell lines expressingspontaneously or after transfection with CLDN18.2. For immunostaining,antibodies reactive to CLDN18.2 can be incubated followed byhorseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbitantibodies according to the vendors instructions.

One particularly preferred methodology for assaying CLDN18.2 in themethods of the invention is Immunohistochemistry or IHC.Immunohistochemistry or IHC refers to the process of detecting antigens(e.g., proteins) in cells of a tissue section, e.g. cells of the tissuesmentioned herein. Immunohistochemical staining is widely used in thediagnosis of abnormal cells such as those found in cancerous tumors.Visualising an antibody-antigen interaction can be accomplished in anumber of ways. In the most common instance, an antibody is conjugatedto an enzyme, such as peroxidase, that can catalyse a colour-producingreaction. Alternatively, the antibody can also be tagged to afluorophore, such as fluorescein or rhodamine.

Preparation of the sample is critical to maintain cell morphology,tissue architecture and the antigenicity of target epitopes. Thisrequires proper tissue collection, fixation and sectioning.Paraformaldehyde is usually used with fixation. Depending on the purposeand the thickness of the experimental sample, either thin (about 4-40μm) sections are sliced from the tissue of interest, or if the tissue isnot very thick and is penetrable it is used whole. The slicing isusually accomplished through the use of a microtome, and slices aremounted on slides.

The sample may require additional steps to make the epitopes availablefor antibody binding, including deparaffinization and antigen retrieval.Detergents like Triton X-100 are generally used in Immunohistochemistryto reduce surface tension, allowing less reagent to be used to achievebetter and more even coverage of the sample.

The direct method of immunohistochemical staining uses one labelledantibody, which binds directly to the antigen being stained for. Theindirect method of immunohistochemical staining which is more commonuses one antibody against the antigen being probed for, and a second,labelled, antibody against the first.

To reduce background staining in IHC, the samples are incubated with abuffer that blocks the reactive sites to which the primary or secondaryantibodies may otherwise bind. Primary antibodies are raised against anantigen of interest and are typically unconjugated (unlabelled), whilesecondary antibodies are raised against immunoglobulins of the primaryantibody species. The secondary antibody is usually conjugated to alinker molecule, such as biotin, that then recruits reporter molecules,or the secondary antibody is directly bound to the reporter moleculeitself. Common blocking buffers include normal serum, non-fat dry milk,BSA or gelatin, and commercial blocking buffers.

Reporter molecules vary based on the nature of the detection method, andthe most popular methods of detection are with enzyme- andfluorophore-mediated chromogenic and fluorescence detection,respectively. With chromogenic reporters, an enzyme label is reactedwith a substrate to yield an intensely colored product that can beanalyzed with an ordinary light microscope. While the list of enzymesubstrates is extensive, alkaline phosphatase (AP) and horseradishperoxidase (HRP) are the two enzymes used most extensively as labels forprotein detection. An array of chromogenic, fluorogenic andchemiluminescent substrates is available for use with either enzyme,including DAB or BCIP/NBT. Fluorescent reporters are small, organicmolecules used for IHC detection. For chromogenic and fluorescentdetection methods, densitometric analysis of the signal can providesemi- and fully-quantitative data, respectively, to correlate the levelof reporter signal to the level of protein expression or localization.

After immunohistochemical staining of the target antigen, a second stainis often applied to provide contrast that helps the primary stain standout. Many of these stains show specificity for discrete cellularcompartments or antigens, while others will stain the whole cell. Bothchromogenic and fluorescent dyes are available for IHC to provide a vastarray of reagents to fit every experimental design. Hematoxylin, Hoechststain and DAPI are commonly used.

Mapping of epitopes recognized by antibodies can be performed asdescribed in detail in “Epitope Mapping Protocols (Methods in MolecularBiology) by Glenn E. Morris ISBN-089603-375-9 and in “Epitope Mapping: APractical Approach” Practical Approach Series, 248 by Olwyn M. R.Westwood, Frank C. Hay.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result in the inhibition of tumor growth and/or inhibitionof tumor development, including inhibition of tumor dissemination andmetastasis. Preferably, immune effector functions result in killing ofcancer cells. Preferably, the immune effector functions in the contextof the present invention are antibody-mediated effector functions. Suchfunctions comprise complement dependent cytotoxicity (CDC),antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependentcell-mediated phagocytosis (ADCP), induction of apoptosis in the cellscarrying the tumor-associated antigen, for example, by binding of theantibody to a surface antigen, inhibition of CD40L-mediated signaltransduction, for example, by binding of the antibody to the CD40receptor or CD40 ligand (CD40L), and/or inhibition of proliferation ofthe cells carrying the tumor-associated antigen, preferably ADCC and/orCDC. Thus, antibodies that are capable of mediating one or more immuneeffector functions are preferably able to mediate killing of cells byinducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypicadhesion, and/or phagocytosis, preferably by inducing CDC-mediated lysisand/or ADCC-mediated lysis. Antibodies may also exert an effect simplyby binding to tumor-associated antigens on the surface of a cancer cell.For example, antibodies may block the function of the tumor-associatedantigen or induce apoptosis just by binding to the tumor-associatedantigen on the surface of a cancer cell.

ADCC describes the cell-killing ability of effector cells, in particularlymphocytes, which preferably requires the target cell being marked byan antibody. ADCC preferably occurs when antibodies bind to antigens oncancer cells and the antibody Fc domains engage Fc receptors (FcR) onthe surface of immune effector cells. Several families of Fc receptorshave been identified, and specific cell populations characteristicallyexpress defined Fc receptors. ADCC can be viewed as a mechanism todirectly induce a variable degree of immediate tumor destruction thatalso leads to antigen presentation and the induction of tumor-directedT-cell responses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and further host-derived antibodyresponses.

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple Clq binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (Clq is one ofthree subcomponents of complement C1). Preferably these uncloaked Clqbinding sites convert the previously low-affinity Clq-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell and may leadto apoptosis.

The term “immune effector cells” in the context of the present inventionrelates to cells which exert effector functions during an immunereaction. For example, such cells secrete cytokines and/or chemokines,kill microbes, secrete antibodies, recognize cancerous cells, andoptionally eliminate such cells. For example, immune effector cellscomprise T-cells (cytotoxic T-cells, helper T-cells, tumor infiltratingT-cells), B-cells, natural killer cells, neutrophils, macrophages, anddendritic cells.

A nucleic acid is according to the invention preferably deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), more preferably RNA, mostpreferably in vitro transcribed RNA (IVT RNA). Nucleic acids includeaccording to the invention genomic DNA, cDNA, mRNA, recombinantlyprepared and chemically synthesized molecules. A nucleic acid mayaccording to the invention be in the form of a molecule which is singlestranded or double stranded and linear or closed covalently to form acircle. A nucleic can be employed for introduction into, i.e.transfection of, cells, for example, in the form of RNA which can beprepared by in vitro transcription from a DNA template. The RNA canmoreover be modified before application by stabilizing sequences,capping, and polyadenylation.

The nucleic acids described herein may be comprised in a vector. Theterm “vector” as used herein includes any vectors known to the skilledperson including plasmid vectors, cosmid vectors, phage vectors such aslambda phage, viral vectors such as adenoviral or baculoviral vectors,or artificial chromosome vectors such as bacterial artificialchromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificialchromosomes (PAC). Said vectors include expression as well as cloningvectors. Expression vectors comprise plasmids as well as viral vectorsand generally contain a desired coding sequence and appropriate DNAsequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments.

As the vector for expression of an antibody, either of a vector type inwhich the antibody chains are present in different vectors or a vectortype in which the antibody chains are present in the same vector can beused.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′-position of a beta-D-ribo-furanose moiety. Theterm includes double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

According to the present invention, the term “RNA” includes andpreferably relates to “mRNA” which means “messenger RNA” and relates toa “transcript” which may be produced using DNA as template and encodes apeptide or protein. mRNA typically comprises a 5′ non translated region,a protein or peptide coding region and a 3′ non translated region. mRNAhas a limited halftime in cells and in vitro.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

Nucleic acids may, according to the invention, be present alone or incombination with other nucleic acids, which may be homologous orheterologous. In preferred embodiments, a nucleic acid is functionallylinked to expression control sequences which may be homologous orheterologous with respect to said nucleic acid. The term “homologous”means that the nucleic acids are also functionally linked naturally andthe term “heterologous” means that the nucleic acids are notfunctionally linked naturally.

A nucleic acid and an expression control sequence are “functionally”linked to one another, if they are covalently linked to one another insuch a way that expression or transcription of said nucleic acid isunder the control or under the influence of said expression controlsequence. If the nucleic acid is to be translated into a functionalprotein, then, with an expression control sequence functionally linkedto a coding sequence, induction of said expression control sequenceresults in transcription of said nucleic acid, without causing a frameshift in the coding sequence or said coding sequence not being capableof being translated into the desired protein or peptide.

The term “expression control sequence” or “expression control element”comprises according to the invention promoters, ribosome binding sites,enhancers and other control elements which regulate transcription of agene or translation of a mRNA. In particular embodiments of theinvention, the expression control sequences can be regulated. The exactstructure of expression control sequences may vary as a function of thespecies or cell type, but generally comprises 5′-untranscribed and 5′-and 3′-untranslated sequences which are involved in initiation oftranscription and translation, respectively, such as TATA box, cappingsequence, CAAT sequence, and the like. More specifically,5′-untranscribed expression control sequences comprise a promoter regionwhich includes a promoter sequence for transcriptional control of thefunctionally linked nucleic acid. Expression control sequences may alsocomprise enhancer sequences or upstream activator sequences.

According to the invention the term “promoter” or “promoter region”relates to a nucleic acid sequence which is located upstream (5′) to thenucleic acid sequence being expressed and controls expression of thesequence by providing a recognition and binding site for RNA-polymerase.The “promoter region” may include further recognition and binding sitesfor further factors which are involved in the regulation oftranscription of a gene. A promoter may control the transcription of aprokaryotic or eukaryotic gene. Furthermore, a promoter may be“inducible” and may initiate transcription in response to an inducingagent or may be “constitutive” if transcription is not controlled by aninducing agent. A gene which is under the control of an induciblepromoter is not expressed or only expressed to a small extent if aninducing agent is absent. In the presence of the inducing agent the geneis switched on or the level of transcription is increased. This ismediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention includepromoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMVpromoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

The term “expression” is used herein in its broadest meaning andcomprises the production of RNA or of RNA and protein or peptide. Withrespect to RNA, the term “expression” or “translation” relates inparticular to the production of peptides or proteins. Expression may betransient or may be stable. According to the invention, the termexpression also includes an “aberrant expression” or “abnormalexpression”.

“Aberrant expression” or “abnormal expression” means according to theinvention that expression is altered, preferably increased, compared toa reference, e.g. a state in a subject not having a disease associatedwith aberrant or abnormal expression of a certain protein, e.g., atumor-associated antigen. An increase in expression refers to anincrease by at least 10%, in particular at least 20%, at least 50% or atleast 100%, or more. In one embodiment, expression is only found in adiseased tissue, while expression in a healthy tissue is repressed.

The term “specifically expressed” means that a protein is essentiallyonly expressed in a specific tissue or organ. For example, atumor-associated antigen specifically expressed in gastric mucosa meansthat said protein is primarily expressed in gastric mucosa and is notexpressed in other tissues or is not expressed to a significant extentin other tissue or organ types. Thus, a protein that is exclusivelyexpressed in cells of the gastric mucosa and to a significantly lesserextent in any other tissue is specifically expressed in cells of thegastric mucosa. In some embodiments, a tumor-associated antigen may alsobe specifically expressed under normal conditions in more than onetissue type or organ, such as in 2 or 3 tissue types or organs, butpreferably in not more than 3 different tissue or organ types. In thiscase, the tumor-associated antigen is then specifically expressed inthese organs.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a protein or peptide.

According to the invention, the term “nucleic acid encoding” means thatnucleic acid, if present in the appropriate environment, preferablywithin a cell, can be expressed to produce a protein or peptide itencodes.

The term “peptide” comprises oligo- and polypeptides and refers tosubstances comprising two or more, preferably 3 or more, preferably 4 ormore, preferably 6 or more, preferably 8 or more, preferably 9 or more,preferably 10 or more, preferably 13 or more, preferably 16 more,preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, inparticular 100 amino acids joined covalently by peptide bonds. The term“protein” refers to large peptides, preferably to peptides with morethan 100 amino acid residues, but in general the terms “peptides” and“proteins” are synonyms and are used interchangeably herein.

Preferably, the proteins and peptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpeptide” mean that the protein or peptide has been separated from itsnatural environment. An isolated protein or peptide may be in anessentially purified state. The term “essentially purified” means thatthe protein or peptide is essentially free of other substances withwhich it is associated in nature or in vivo.

The teaching given herein with respect to specific amino acid sequences,e.g. those shown in the sequence listing, is to be construed so as toalso relate to modifications, i.e. variants, of said specific sequencesresulting in sequences which are functionally equivalent to saidspecific sequences, e.g. amino acid sequences exhibiting propertiesidentical or similar to those of the specific amino acid sequences. Oneimportant property is to retain binding of an antibody to its target.Preferably, a sequence modified with respect to a specific sequence,when it replaces the specific sequence in an antibody retains binding ofsaid antibody to the target.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR sequences, hypervariable and variable regionscan be modified without losing the ability to bind to a target. Forexample, CDR sequences will be either identical or highly homologous tothe CDR sequences specified herein.

By “highly homologous” it is contemplated that from 1 to 5, preferablyfrom 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made.

The term “variant” according to the invention also includes mutants,splice variants, conformations, isoforms, allelic variants, speciesvariants and species homologs, in particular those which are naturallypresent. An allelic variant relates to an alteration in the normalsequence of a gene, the significance of which is often unclear. Completegene sequencing often identifies numerous allelic variants for a givengene. A species homolog is a nucleic acid or amino acid sequence with adifferent species of origin from that of a given nucleic acid or aminoacid sequence.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants. Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

Homologous amino acid sequences exhibit according to the invention atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98 or atleast 99% identity of the amino acid residues.

The amino acid sequence variants described herein may readily beprepared by the skilled person, for example, by recombinant DNAmanipulation. The manipulation of DNA sequences for preparing proteinsand peptides having substitutions, additions, insertions or deletions,is described in detail in Sambrook et al. (1989), for example.Furthermore, the peptides and amino acid variants described herein maybe readily prepared with the aid of known peptide synthesis techniquessuch as, for example, by solid phase synthesis and similar methods.

The invention includes derivatives of the peptides or proteins describedherein which are comprised by the terms “peptide” and “protein”.According to the invention, “derivatives” of proteins and peptides aremodified forms of proteins and peptides. Such modifications include anychemical modification and comprise single or multiple substitutions,deletions and/or additions of any molecules associated with the proteinor peptide, such as carbohydrates, lipids and/or proteins or peptides.In one embodiment, “derivatives” of proteins or peptides include thosemodified analogs resulting from glycosylation, acetylation,phosphorylation, amidation, palmitoylation, myristoylation,isoprenylation, lipidation, alkylation, derivatization, introduction ofprotective/blocking groups, proteolytic cleavage or binding to anantibody or to another cellular ligand. The term “derivative” alsoextends to all functional chemical equivalents of said proteins andpeptides. Preferably, a modified peptide has increased stability and/orincreased immunogenicity.

According to the invention, a variant, derivative, modified form,fragment, part or portion of an amino acid sequence, peptide or proteinpreferably has a functional property of the amino acid sequence, peptideor protein, respectively, from which it has been derived, i.e. it isfunctionally equivalent. In one embodiment, a variant, derivative,modified form, fragment, part or portion of an amino acid sequence,peptide or protein is immunologically equivalent to the amino acidsequence, peptide or protein, respectively, from which it has beenderived. In one embodiment, the functional property is an immunologicalproperty.

The term “derived” means according to the invention that a particularentity, in particular a particular sequence, is present in the objectfrom which it is derived, in particular an organism or molecule. In thecase of amino acid sequences, especially particular sequence regions,“derived” in particular means that the relevant amino acid sequence isderived from an amino acid sequence in which it is present.

The term “cell” or “host cell” preferably is an intact cell, i.e. a cellwith an intact membrane that has not released its normal intracellularcomponents such as enzymes, organelles, or genetic material. An intactcell preferably is a viable cell, i.e. a living cell capable of carryingout its normal metabolic functions. The term “cell” includes accordingto the invention prokaryotic cells (e.g., E. coli) or eukaryotic cells(e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells,HEK293 cells, HELA cells, yeast cells, and insect cells). Mammaliancells are particularly preferred, such as cells from humans, mice,hamsters, pigs, goats, and primates. The cells may be derived from alarge number of tissue types and include primary cells and cell lines.The term “cell” includes non-cancerous cells and cancer cells such ascells of the cancer types disclosed herein.

A cell which comprises a nucleic acid molecule preferably express thepeptide or protein encoded by the nucleic acid.

“Target cell” shall mean a cell which is a target for an immune responsesuch as an antibody. Target cells include any undesirable cell such as acancer cell as described herein. In preferred embodiments, the targetcell is a cell expressing CLDN18.2. Cells expressing CLDN18.2 typicallyinclude cancer cells.

The terms “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably antibody heavy and/orlight chain transgenes, or transchromosomes (either integrated ornon-integrated into the animal's natural genomic DNA) and which ispreferably capable of expressing the transgenes. For example, atransgenic mouse can have a human light chain transgene and either ahuman heavy chain transgene or human heavy chain transchromosome, suchthat the mouse produces human anti-CLDN18.2 antibodies when immunizedwith CLDN18.2 antigen and/or cells expressing CLDN18.2. The human heavychain transgene can be integrated into the chromosomal DNA of the mouse,as is the case for transgenic mice, e.g., HuMAb mice, such as HCo7 orHCo12 mice, or the human heavy chain transgene can be maintainedextrachromosomally, as is the case for transchromosomal (e.g., KM) miceas described in WO 02/43478. Such transgenic and transchromosomal micemay be capable of producing multiple isotypes of human monoclonalantibodies to CLDN18.2 (e.g., IgG, IgA and/or IgE) by undergoing V-D-Jrecombination and isotype switching.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral immune reaction, the strength and/or durationof the induced immune reaction, or the specificity of the immunereaction. In the context of the present invention, the term“immunologically equivalent” is preferably used with respect to theimmunological effects or properties of a peptide or peptide variant usedfor immunization or an antibody. A particular immunological property isthe ability to bind to antibodies and, where appropriate, generate animmune response, preferably by stimulating the generation of antibodies.For example, an amino acid sequence is immunologically equivalent to areference amino acid sequence if said amino acid sequence when exposedto the immune system of a subject induces an immune reaction, preferablyantibodies, having a specificity of reacting with the reference aminoacid sequence, such as the reference amino acid sequence forming part ofCLDN18.2.

The invention provides methods for detecting the presence of CLDN18.2antigen in a sample, or measuring the amount of CLDN18.2 antigen,comprising contacting the sample, and optionally a control sample, withan antibody of the invention which binds to CLDN18.2, under conditionsthat allow for formation of a complex between the antibody and CLDN18.2.The formation of a complex is then detected, wherein a difference incomplex formation between the sample compared to a control sample isindicative for the presence of CLDN18.2 antigen in the sample.

Methods as described above are useful, in particular, for diagnosingCLDN18.2-related diseases such as cancer diseases. Preferably an amountof CLDN18.2 in a sample which is higher than the amount of CLDN18.2 in areference or control sample is indicative for the presence of aCLDN18.2-related disease in a subject, in particular a human, from whichthe sample is derived.

When used in methods as described above, an antibody described hereinmay be provided with a label that functions to: (i) provide a detectablesignal; (ii) interact with a second label to modify the detectablesignal provided by the first or second label, e.g. FRET (FluorescenceResonance Energy Transfer); (iii) affect mobility, e.g. electrophoreticmobility, by charge, hydrophobicity, shape, or other physicalparameters, or (iv) provide a capture moiety, e.g., affinity,antibody/antigen, or ionic complexation. Suitable as label arestructures, such as fluorescent labels, luminescent labels, chromophorelabels, radioisotopic labels, isotopic labels, preferably stableisotopic labels, isobaric labels, enzyme labels, particle labels, inparticular metal particle labels, magnetic particle labels, polymerparticle labels, small organic molecules such as biotin, ligands ofreceptors or binding molecules such as cell adhesion proteins orlectins, label-sequences comprising nucleic acids and/or amino acidresidues which can be detected by use of binding agents, etc. Labelscomprise, in a nonlimiting manner, barium sulfate, iocetamic acid,iopanoic acid, calcium ipodate, sodium diatrizoate, megluminediatrizoate, metrizamide, sodium tyropanoate and radio diagnostic,including positron emitters such as fluorine-18 and carbon-11, gammaemitters such as iodine-123, technetium-99m, iodine-131 and indium-111,nuclides for nuclear magnetic resonance, such as fluorine andgadolinium.

According to the invention, a “reference” such as a reference sample orreference organism may be used to correlate and compare the resultsobtained in the methods of the invention from a test sample or testorganism. Typically the reference organism is a healthy organism, inparticular an organism which does not suffer from a disease such as acancer disease. A “reference value” or “reference level” can bedetermined from a reference empirically by measuring a sufficientlylarge number of references. Preferably the reference value is determinedby measuring at least 2, preferably at least 3, preferably at least 5,preferably at least 8, preferably at least 12, preferably at least 20,preferably at least 30, preferably at least 50, or preferably at least100 references.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level. The term “inhibit” or similar phrases includesa complete or essentially complete inhibition, i.e. a reduction to zeroor essentially to zero.

Terms such as “increase” or “enhance” preferably relate to an increaseor enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%.

The agents, compositions and methods described herein can be used todiagnose a subject with a disease. Diseases which can be diagnosedencompass all diseases expressing CLDN18.2. Particularly preferreddiseases are cancer diseases such as cancer diseases described herein.

According to the invention, the term “disease” refers to anypathological state, including cancer diseases, in particular those formsof cancer diseases described herein.

The term “normal” such as used in the terms “normal tissue” or “normalconditions” refers to healthy tissue or the conditions in a healthysubject, i.e., non-pathological conditions, wherein “healthy” preferablymeans non-cancerous.

“Disease involving cells expressing CLDN18.2” means according to theinvention that expression of CLDN18.2 in cells of a diseased tissue ororgan is preferably increased compared to the state in a healthy tissueor organ. An increase refers to an increase by at least 10%, inparticular at least 20%, at least 50%, at least 100%, at least 200%, atleast 500%, at least 1000%, at least 10000% or even more. In oneembodiment, expression is only found in a diseased tissue, whileexpression in a healthy tissue is repressed. According to the invention,diseases involving or being associated with cells expressing CLDN18.2include cancer diseases, in particular those forms of cancer describedherein.

According to the invention, the term “tumor” or “tumor disease” refersto a swelling or lesion formed by an abnormal growth of cells (calledneoplastic cells or tumor cells). By “tumor cell” is meant an abnormalcell that grows by a rapid, uncontrolled cellular proliferation andcontinues to grow after the stimuli that initiated the new growth cease.Tumors show partial or complete lack of structural organization andfunctional coordination with the normal tissue, and usually form adistinct mass of tissue, which may be either benign, pre-malignant ormalignant.

A benign tumor is a tumor that lacks all three of the malignantproperties of a cancer. Thus, by definition, a benign tumor does notgrow in an unlimited, aggressive manner, does not invade surroundingtissues, and does not spread to non-adjacent tissues (metastasize).Common examples of benign tumors include moles and uterine fibroids.

The term “benign” implies a mild and nonprogressive disease, and indeed,many kinds of benign tumors are harmless to the health. However, someneoplasms which are defined as “benign tumors” because they lack theinvasive properties of a cancer, may still produce negative healtheffects. Examples of this include tumors which produce a “mass effect”(compression of vital organs such as blood vessels), or “functional”tumors of endocrine tissues, which may overproduce certain hormones(examples include thyroid adenomas, adrenocortical adenomas, andpituitary adenomas).

Benign tumors typically are surrounded by an outer surface that inhibitstheir ability to behave in a malignant manner. In some cases, certain“benign” tumors may later give rise to malignant cancers, which resultfrom additional genetic changes in a subpopulation of the tumor'sneoplastic cells. A prominent example of this phenomenon is the tubularadenoma, a common type of colon polyp which is an important precursor tocolon cancer. The cells in tubular adenomas, like most tumors whichfrequently progress to cancer, show certain abnormalities of cellmaturation and appearance collectively known as dysplasia. Thesecellular abnormalities are not seen in benign tumors that rarely ornever turn cancerous, but are seen in other pre-cancerous tissueabnormalities which do not form discrete masses, such as pre-cancerouslesions of the uterine cervix. Some authorities prefer to refer todysplastic tumors as “pre-malignant”, and reserve the term “benign” fortumors which rarely or never give rise to cancer.

Neoplasm is an abnormal mass of tissue as a result of neoplasia.Neoplasia (new growth in Greek) is the abnormal proliferation of cells.The growth of the cells exceeds, and is uncoordinated with that of thenormal tissues around it. The growth persists in the same excessivemanner even after cessation of the stimuli. It usually causes a lump ortumor. Neoplasms may be benign, pre-malignant or malignant.

“Growth of a tumor” or “tumor growth” according to the invention relatesto the tendency of a tumor to increase its size and/or to the tendencyof tumor cells to proliferate.

Cancer (medical term: malignant neoplasm) is a class of diseases inwhich a group of cells display uncontrolled growth (division beyond thenormal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis (spread to other locations in thebody via lymph or blood). These three malignant properties of cancersdifferentiate them from benign tumors, which are self-limited, and donot invade or metastasize. Most cancers form a tumor but some, likeleukemia, do not. According to the invention, the terms “cancer” and“tumor” or “cancer disease” and “tumor disease” are generally usedinterchangeably herein to refer to diseases wherein cells display anuncontrolled growth and optionally invasion and/or metastasis.

Preferably, a “cancer disease” according to the invention ischaracterized by cells expressing CLDN18.2. A cell expressing CLDN18.2preferably is a cancer cell, preferably of the tumors and cancersdescribed herein. Preferably, such cell is a cell other than a stomachcell.

Cancers are classified by the type of cell that resembles the tumor and,therefore, the tissue presumed to be the origin of the tumor. These arethe histology and the location, respectively.

The term “cancer” according to the invention comprises leukemias,seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas,rectal cancer, endometrial cancer, kidney cancer, adrenal cancer,thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervicalcancer, intestinal cancer, liver cancer, colon cancer, stomach cancer,intestine cancer, head and neck cancer, gastrointestinal cancer, lymphnode cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear,nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer ofthe uterus, ovarian cancer and lung cancer and the metastases thereof.Examples thereof are lung carcinomas, mamma carcinomas, prostatecarcinomas, colon carcinomas, renal cell carcinomas, cervicalcarcinomas, or metastases of the cancer types or tumors described above.The term cancer according to the invention also comprises cancermetastases.

The main types of lung cancer are small cell lung carcinoma (SCLC) andnon-small cell lung carcinoma (NSCLC). There are three main sub-types ofthe non-small cell lung carcinomas: squamous cell lung carcinoma,adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas accountfor approximately 10% of lung cancers. This cancer usually is seenperipherally in the lungs, as opposed to small cell lung cancer andsquamous cell lung cancer, which both tend to be more centrally located.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells. This group represents the most common cancers,including the common forms of breast, prostate, lung and colon cancer.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor, i.e. a secondary tumor ormetastatic tumor, at the target site depends on angiogenesis. Tumormetastasis often occurs even after the removal of the primary tumorbecause tumor cells or components may remain and develop metastaticpotential. In one embodiment, the term “metastasis” according to theinvention relates to “distant metastasis” which relates to a metastasiswhich is remote from the primary tumor and the regional lymph nodesystem.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if ovarian cancermetastasizes to the liver, the secondary tumor is made up of abnormalovarian cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic ovarian cancer, not liver cancer.

A relapse or recurrence occurs when a person is affected again by acondition that affected them in the past. For example, if a patient hassuffered from a cancer disease, has received a successful treatment ofsaid disease and again develops said disease said newly developeddisease may be considered as relapse or recurrence. However, accordingto the invention, a relapse or recurrence of a cancer disease may butdoes not necessarily occur at the site of the original cancer disease.Thus, for example, if a patient has suffered from ovarian tumor and hasreceived a successful treatment a relapse or recurrence may be theoccurrence of an ovarian tumor or the occurrence of a tumor at a sitedifferent to ovary. A relapse or recurrence of a tumor also includessituations wherein a tumor occurs at a site different to the site of theoriginal tumor as well as at the site of the original tumor. Preferably,the original tumor for which the patient has received a treatment is aprimary tumor and the tumor at a site different to the site of theoriginal tumor is a secondary or metastatic tumor.

By “treat” is meant to administer a compound or composition to a subjectin order to prevent or eliminate a disease, including reducing the sizeof a tumor or the number of tumors in a subject; arrest or slow adisease in a subject; inhibit or slow the development of a new diseasein a subject; decrease the frequency or severity of symptoms and/orrecurrences in a subject who currently has or who previously has had adisease; and/or prolong, i.e. increase the lifespan of the subject.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, preventing, slowing down orinhibiting progression or worsening, or preventing or delaying the onsetof a disease or the symptoms thereof.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing adisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a disease, inparticular cancer is a subject who has an increased risk for developinga disease, as such a subject may continue to develop a disease. Subjectswho currently have, or who have had, a cancer also have an increasedrisk for cancer metastases.

The term “immunotherapy” relates to a treatment involving a specificimmune reaction. In the context of the present invention, terms such as“protect”, “prevent”, “prophylactic”, “preventive”, or “protective”relate to the prevention or treatment or both of the occurrence and/orthe propagation of a disease in a subject and, in particular, tominimizing the chance that a subject will develop a disease or todelaying the development of a disease. For example, a person at risk fora tumor, as described above, would be a candidate for therapy to preventa tumor. Immunotherapy may be performed using any of a variety oftechniques, in which agents function to remove antigen-expressing cellsfrom a patient.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against diseased cells with theadministration of immune response-modifying agents (such asimmunoreactive peptides and nucleic acids).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as antibodies) that can directly orindirectly mediate antitumor effects and does not necessarily depend onan intact host immune system.

The term “in vivo” relates to the situation in a subject.

The terms “subject”, “individual”, “organism” or “patient” are usedinterchangeably and relate to vertebrates, preferably mammals. Forexample, mammals in the context of the present invention are humans,non-human primates, domesticated animals such as dogs, cats, sheep,cattle, goats, pigs, horses etc., laboratory animals such as mice, rats,rabbits, guinea pigs, etc. as well as animals in captivity such asanimals of zoos. The term “animal” as used herein also includes humans.The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease, preferably a disease as describedherein.

According to the invention, a “sample” may be any sample usefulaccording to the present invention, in particular a biological samplesuch a tissue sample, including body fluids, and/or a cellular sampleand may be obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“sample” also includes processed samples such as fractions or isolatesof biological samples, e.g. nucleic acid and peptide/protein isolates.Preferably a sample contains cells or tissue of the organ which is to beexamined, e.g. which is to be diagnosed for cancer. For example, if thecancer to be diagnosed is lung cancer a sample may contain cells ortissue obtained from lung.

According to the invention a sample may be a sample such as a bodilysample derived from a patient containing or being expected of containingtumor or cancer cells. The bodily sample may be any tissue sample suchas blood, a tissue sample obtained from the primary tumor or from tumormetastases or any other sample containing tumor or cancer cells.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methodsincluding the use of kits and reagents are carried out according to themanufacturers' information unless specifically indicated.

Example 1: Generation of Monoclonal Antibodies

The aim of this project was to generate murine monoclonalCLDN18-specific antibodies capable of detecting CLDN18.2 expressingtumor cells in stomach CA, esophagus CA, pancreas CA and lung CA FFPEtissues.

To generate a highly specific, high affinity diagnostic CLDN18.2antibody it was essential to start immunization protocols with a bigvariation of different immunogens and adjuvants. During the projectabout 100 mice (C57Bl/6 and Balb/c) were inoculated, using variousimmunization strategies to trigger an α-CLDN18 immune response.

To trigger the mouse immune system and to overcome the immune tolerancewe used virus-like-particles (VLP), peptide-conjugates and recombinantproteins coding for different parts of human CLDN18.2 expressed asrecombinant fusion proteins with different expression partners (tags).

Out of 13 different immunization strategies the best results wereachieved by treating mice with HIS-tagged CLDN18 C-termal recombinantprotein (see FIG. 2; Immunization #20) in combination with variousadjuvants (see Table 1, below, fusion 35).

One candidate (35-22A) resulted from a 4 step-immunization strategy (30days). A further candidate (43-14A) was generated following a7-step-immunization protocol (79 days)(see Table 1, below, fusion 43).

Two days before splenectomy, the mice were boosted to activate thetargeted B-cells.

On the day of fusion the mouse splenocytes were isolated and fused to amouse myeloma cell line Ag8.653. For fusion of mouse cells to themyeloma we followed the standard protocol published by Köhler andMilstein 1975. After HAT selection supernatants were tested in ELISA forsecretion of antibodies recognizing the antigen used for immunizations.

The hybridoma cells of ELISA positive supernatants were subcloned togenerate monoclonal hybridomas and supernatants of the subclonedhybridoma cells were rescreened in ELISA. Hybridoma cells of positiveclones were expanded and supernatants analyzed further.

Example 2: Western Blot Screen of Monoclonal Hybridoma Supernatants

To answer the question if ELISA-positive antibodies in the supernatantsare able to bind to either recombinant claudin 18 or protein lysatesfrom stable transfected claudin 18 expressing HEK293 cells WesternBlot-analysis was performed. Antibodies which were able to bindspecifically to claudin 18 in a Western Blot-analysis were expanded.Cells were cryoconserved and antibodies purified via MABselect (FPLC).The antibodies selected by the Western Blot screening were purified andevaluated for their ability to bind their antigen in formalin fixedparaffin-embedded tissues (FFPE) by immunohistochemistry.

Example 3: Histological Analysis—First Screen of Western Blot PositiveAntibodies

The aim of this experiment was to check the CLDN18 specificity andsensitivity of the antibodies. This was done by using CLDN18 expressingFFPE normal stomach tissue.

In a first experiment the Western Blot tested purified antibodies wereanalyzed at a concentration of 0.5 μg/ml on human stomach FFPE sections.Antibodies which performed well and did not produce high amounts ofbackground were further titrated to 0.2 & 0.1 μg/ml on various normalstomach tissues to test the sensitivity and specificity. In laterdevelopment stages the freshly generated antibodies were directly testedat a concentration of 0.2 μg/ml because the best antibody alreadyperformed very well at 0.2 μg/ml and did serve as a benchmark.Antibodies generating strong signals on the mucosa epithelium of thetested human stomach tissues and no background on the adjacent mucosatissues were selected for further titration experiments and specificityanalysis. Two antibodies did perform outstanding: 35-22A and 43-14A; seeTables 2 and 3, below.

Antibodies producing strong signals on the tested normal stomach tissuewere further analyzed on cancer tissues. The corresponding hybridomacells were adapted to serum free media. The signals produced using mumAb43-14A were slightly stronger than the signals produced using mumAb35-22A; see Table 4, below.

Example 4: Histological in Depth Analysis and Antibody Characterization

The serum free produced antibodies were used to stain stomach CA tissuemicroarrays (TMA). The amount of stained cases, the strength of thesignal and the amount of positive tumor cells were analyzed.

The staining intensities of the mumAbs 35-22A and 43-14A were excellent.No significant differences in the staining pattern and only slightdifferences in the staining intensities between the tested antibodies35-22A and 43-14A were detected.

Example 5: Analysis of Antibody Specificity Using a Normal Tissue Panel

The selected antibodies were tested on various, relevant normal tissuesto ensure the high CLDN18 target specificity; see Tables 5A and 5B,below.

No significant differences in the staining pattern and stainingintensities of the antibodies 35-22A and 43-14A were visible in theprevious experiments. Therefore the antibodies were subjected tostaining experiments with a more clinically oriented protocol. Tosimulate the staining processes applied in standard pathology labs aOne-Day-Protocol with a short (1 hour) primary antibody incubation stepwas established.

In all analyzed cases mumAb 43-14A performed extremely good and evenbetter compared to mumAb 35-22A; see Table 6, below.

Example 6: In Depth Analysis on Relevant Tissue—Respiratory Epithelium

mumAb 43-14A was additionally analyzed on various relevant respiratorytissues to ensure its specificity, especially in target tissues of thelung/bronchial tract. For these tissues the expression of CLDN18.1 wasreported. To analyze whether the diagnostic antibody cross reacts withthe lung/bronchial expressed isoform of CLDN18.1 all availablelung/bronchial tissues were screened. No signals were detected with lungand bronchial tissues; see Table 7, below. The CLDN18 isoform expressedin these respiratory tissues is not recognized by the antibody 43-14A.

Example 7: Epitope Mapping of the mumAbs 43-14A and 35-22A

Peptide ELISA was performed to identify the antibody-binding epitopes onCLDN18.2. Each purified antibody was tested on overlapping peptidescovering the C-terminal sequence of CLDN18.2. 35-22A and 43-14A bothshowed specific binding to an epitope mapping to the peptideTEDEVQSYPSKHDYV. The following sequence was determined as the reactivesequence: EVQSYPSKHDYV.

Example 8: Sequence Analysis of the mumAbs 43-14A and 35-22A

An analysis of the sequence of the antibodies 43-14A and 35-22A is shownin FIG. 3.

Example 9: Staining of Different Cancer Tissues

Immunohistochemistry (IHC) was performed on slides of 4% bufferedformalin fixed paraffin embedded samples. Paraffin embedding wasperformed according to standard protocols.

After deparaffinization, all slides were subjected to antigen retrievalby boiling in 10 mM citric acid supplemented with 0.05% Tween-20 (pH6.0) at 120° C. for 10 min, subsequently quenched (by 2% H2O2) blockedand incubated overnight at 4° C. with 0.2 to 0.5 μg/ml diagnosticmonoclonal mouse anti-CLNDN18.2 antibody 43-14A or 35-22A. Antibodybinding was visualized with horseradish-peroxidase-labeled secondaryantibodies using the polymer-based Powervision antibody (Power VisionHRP goat-a-mouse; Immunologic, Duiven, The Netherlands) and asubstrate-chromogen solution (VectorRed; Vector Labs, Burlingame, USA).Sections were subsequently counter-stained with Mayer's haematoxylin(Carl Roth GmbH, Karlsruhe, Germany) and subjected to evaluation by theraters.

Histological Assessment

All samples were analyzed regarding the relative proportion of positivestained tumor cells in relation to all visible tumor cells for eachsection. The intensity of the staining was classified as negative (−),weakly positive (1+), medium positive (2+) and strongly positive (3+).Only membranous staining was considered as positive. Human stomachtissue served as positive control for each staining. Since PanIN(pancreatic intraepithelial neoplasia) are frequently found strongpositive, those areas were also considered as internal stainingintensity reference for strong positivity (3+).

Strong, membranous signals were generated by both antibodies inpancreatic, esophageal and stomach cancerous tissues (Table 8) or withantibody 43-14A in lung cancerous tissues (Table 9). The number ofpositive tumor cells varied interindividually between the differenttumor cases. The biggest part of the analysed samples was 2+ to 3+positive.

TABLE 1 Immunization schemes for antibodies Mouse Antigen AdjuvantAdministration Date Day Event Strain ID [μl] [μg] Code [μl]/[μg] CodeRoute Volume Mouse 5 Immunization #20 - Fusion 35 27. Oct. 2010 0 1.Immunization C57BL/6 M5 100 100 C-terminal 100 Gerbu MM i.p. 200 μlGC182-HIS 4. Nov. 2010 7 2. Immunization C57BL/6 M5 100 100 C-terminal50/50 CpG-PTO i.p. 200 μl GC182-HIS 10. Nov. 2010 14 3. ImmunizationC57BL/6 M5 100 100 C-terminal 100 Gerbu MM i.p. 200 μl GC182-HIS 17.Nov. 2010 21 4. Immunization C57BL/6 M5 100 100 C-terminal 100 Gerbu MMi.p. 200 μl GC182-HIS 24. Nov. 2010 28 Boost C57BL/6 M5 100 100C-terminal 100 Gerbu MM i.p. 200 μl GC182-HIS 26. Nov. 2010 30 Fusion#35 C57BL/6 M5 Mouse 4 Immunization #20 - Fusion 43 27. Oct. 2010 0 1.Immunization BALB/c M4 100 100 C-terminal 100 Gerbu MM i.p. 200 μlGC182-HIS 10. Nov. 2010 14 2. Immunization BALB/c M4 100 100 C-terminal100 Gerbu MM i.p. 200 μl GC182-HIS 17. Nov. 2010 21 3. ImmunizationBALB/c M4 100 100 C-terminal 100 Gerbu MM i.p. 200 μl GC182-HIS 24. Nov.2010 28 4. Immunization BALB/c M4 100 100 C-terminal 100 Gerbu MM i.p.200 μl GC182-HIS 8. Dec. 2010 42 5. Immunization BALB/c M4 100 100C-terminal 100 Gerbu MM i.p. 200 μl GC182-HIS 22. Dec. 2010 56 6.Immunization BALB/c M4 100 100 C-terminal 100 Gerbu MM i.p. 200 μlGC182-HIS 5. Jan. 2011 70 7. Immunization BALB/c M4 100 100 C-terminal100 Gerbu MM i.p. 200 μl GC182-HIS 12. Jan. 2011 77 Boost BALB/c M4 100100 C-terminal 100 Gerbu MM i.p. 200 μl GC182-HIS 14. Jan. 2011 79Fusion #43 BALB/c M4

TABLE 2 mumAbs positive selected by Western Blot analysis AntibodyCryo-/ Mucosa Subcellular % positive bg on lamina smooth slide numberTissue Antibody conc. Paraffin epithelium pattern cells propriaLymphocytes Vessels Musculature 11_413 human 35-22A 0.5 μg/ml Paraffin+++ m 90 − − − − 11_910 human 43-14A 0.2 μg/ml Paraffin +++ m 90 − − − −

TABLE 3 Comparison of the two antibodies 35-22A and 43-14A on normalhuman stomach FFPE tissue % bg & Mucosa Subcel- posi- cells in Fi-smooth Tissue Anti- Antibody devel- epithe- lular tive lamina Lympho-brous Ves- Muscu- Slide ID detail body conc. opment lium pattern cellspropria cytes tissue sels lature Comment 11_413 stomach 5 35-22A 0.5μg/ml 1:30 min +++ m 90 − − − − − very strong membranous staining of themucosa epithelium, no bg on stroma, vessels and muscles The 43-14Awasn't tested at 0.5 μg/ml, because the 35-22A did already perform verywell at 0.2 μg/ml and did serve as a rule. 11_975 stomach 1 35-22A 0.1μg/ml 2:30 min +++ m 90 − − − − − strong membranous staining of themucosa epithelium, no bg on stroma, muscles, vessels or lamina propria11_975 stomach 1 43-14A 0.1 μg/ml 2:30 min +++ m 90 − − − − − strongmembranous staining of the mucosa epithelium, no bg on stroma, muscles,vessels or lamina propria 11_907 stomach 5 35-22A 0.2 μg/ml 2:30 min +++m 90 − − − − − strong membranous staining of the mucosa epithelium, nobg on stroma, muscles, vessels or lamina propria 11_910 stomach 5 43-14A0.2 μg/ml 2:30 min +++ m 90 − − − − − strong membranous staining of themucosa epithelium, no bg on stroma, muscles, vessels or lamina propria

TABLE 4 Cancer tissue analysis - TMA127A Cryo-/ slide number Tissuedetail Tissue id Antibody Antibody conc. Paraffin development 11_474Stomach CA (+++) B04/01221 II 35-22A 0.1 μg/ml Paraffin 2:30 min 11_474Stomach CA (++) B06/09514 (5 35-22A 0.1 μg/ml Paraffin 2:30 min 11_474Stomach CA (+) B05/09809 (4 35-22A 0.1 μg/ml Paraffin 2:30 min 11_474Renal CA B08/13471 (3 35-22A 0.1 μg/ml Paraffin 2:30 min 11_475 StomachCA (+++) B04/01221 II 43-14A 0.1 μg/ml Paraffin 2:30 min 11_475 StomachCA (++) B06/09514 (5 43-14A 0.1 μg/ml Paraffin 2:30 min 11_475 StomachCA (+) B05/09809 (4 43-14A 0.1 μg/ml Paraffin 2:30 min 11_475 Renal CAB08/13471 (3 43-14A 0.1 μg/ml Paraffin 2:30 min Normal Subcellular %positive epithelial smooth slide number Tumor cells pattern cells cellsFibrous tissue Vessels Musculature 11_474 ++/+++ m 90 n.a. − − − 11_474(+)/+ c/m 10 n.a. − − − 11_474 − n.a. 0 n.a. − − − 11_474 − n.a. 0 n.a.− − n.a. 11_475 +++ m 90 n.a. − − − 11_475 (+)/+ m/c 10 n.a. − − −11_475 − − 0 n.a. − − − 11_475 − n.a. 0 n.a. − − n.a.

TABLE 5A Normal tissue analysis Normal epithelial cells and Subcel- %Fi- smooth slide Tissue Anti- Antibody Cryo-/ devel- functional lularpositive Lympho- brous Ves- Muscu- Fatty number Tissue id body conc.Paraffin opment tissue pattern cells cytes tissue sels lature tissue11_577 Stomach Stomach 9 35-22A 0.2 μg/ml Paraffin 00:50 ++/+++ m 90 − −− − n.a. 11_1756 Stomach Stomach 9 43-14A 0.2 μg/ml Paraffin 00:50 +++m >90 − − − − n.a. 11_580 Colon Colon 2 35-22A 0.2 μg/ml Paraffin 02:15− n.a. n.a. − − − − n.a. 11_1753 Colon Colon 2 43-14A 0.2 μg/ml Paraffin02:15 − n.a. n.a. − − − − − 11_586 Kidney Kidney 2 35-22A 0.2 μg/mlParaffin 02:15 − n.a. n.a. − − − − n.a. 11_1754 Kidney Kidney 2 43-14A0.2 μg/ml Paraffin 02:15 − n.a. n.a. n.a. − − − − 11_589 Lung Lung 235-22A 0.2 μg/ml Paraffin 02:15 − n.a. n.a. − − − − n.a. 11_1663 LungLung 2 43-14A 0.2 μg/ml Paraffin 03:00 − n.a. n.a. − − − − n.a. 11_595Pancreas Pancreas 3 35-22A 0.2 μg/ml Paraffin 02:15 − n.a. n.a. − − − −− 11_1749 Pancreas Pancreas3 43-14A 0.2 μg/ml Paraffin 02:15 − n.a. n.a.n.a. − − − − 11_599 Liver Liver 1 35-22A 0.2 μg/ml Paraffin 02:15 − n.a.n.a. − − − − − 11_1751 Liver Liver 1 43-14A 0.2 μg/ml Paraffin 02:15 −n.a. n.a. n.a. − − − −

TABLE 5B Normal tissue analysis - 43-14 A Normal epithelial cells andSubcel- % Fi- smooth slide Tissue Anti- Antibody Cryo-/ devel-functional lular positive Lympho- brous Ves- Muscu- Fatty number Tissueid body conc. Paraffin opment tissue pattern cells cytes tissue selslature tissue 11_1748 Pancreas Pancreas 5 43-14A 0.2 μg/ml Paraffin02:15 − n.a. n.a. n.a. − − − − SF 11_1750 Liver Liver 4.5 43-14A 0.2μg/ml Paraffin 02:15 − n.a. n.a. n.a. − − − − SF 11_1755 Kidney Kidney 343-14A 0.2 μg/ml Paraffin 02:15 − n.a. n.a. n.a. − − − − SF 11_1757Stomach Stomach 43-14A 0.2 μg/ml Paraffin 02:15 +++ m >90 − − − − n.a.12 SF 11_1758 Heart Heart 1 43-14A 0.2 μg/ml Paraffin 02:15 − n.a. n.a.n.a. − − − − SF 11_1759 Heart Heart 2 43-14A 0.2 μg/ml Paraffin 02:15 −n.a. n.a. n.a. − − − − SF

TABLE 6 Normal tissue analysis - One-Day-Protocol func- Subcel- % Lym-Fi- smooth slide Antibody devel- tional tumor lular positive pho- brousVes- Muscu- Fatty number Tissue Tissue id Antibody conc. opment tissuetissue pattern cells cytes tissue sels lature tissue 11_2092 StomachStomach 9 43-14 A 0.1 μg/ml 03:30 ++/+++ n.a. m >90 − − − − − 11_2092Stomach Stomach 9 35-22 A 0.1 μg/ml 03:30 +/++ n.a. m >90 − − − − −11_2093 Stomach Stomach 9 43-14 A SF 0.1 μg/ml 03:30 +++ n.a. m >90 − −− − − 11_2093 Stomach Stomach 9 35-22 A SF 0.1 μg/ml 03:30 + n.a. m70-80 − − − − − 11_2094 TMA 127 A TMA 127 A 43-14 A 0.1 μg/ml 03:30 n.a.+/++ m 90 − − − n.a. n.a. 11_2094 TMA 127 A TMA 127 A 35-22 A 0.1 μg/ml03:30 n.a. + m <5 − − − n.a. n.a. 11_2095 TMA 127 A TMA 127 A 43-14 A SF0.1 μg/ml 03:30 n.a. +/++ m 90 − − − n.a. n.a. 11_2095 TMA 127 A TMA 127A 35-22 A SF 0.1 μg/ml 03:30 n.a. −/(+) c <5 − − − n.a. n.a.

TABLE 7 Analysis of normal respiratory tissues Normal % slide TissueAntibody epithelial Subcellular positive Fibrous smooth number idAntibody conc. cells pattern cells Lymphocytes tissue VesselsMusculature 11_1660 lung 1 muAb 43-14A 0.2 μg/ml − − − n.a. − − −11_1660 lung 1 muAb 43-14A 0.5 μg/ml − − − n.a. − − − 11_1663 lung 2muAb 43-14A 0.2 μg/ml − − − − − − − 11_1663 lung 2 muAb 43-14A 0.5 μg/ml− − − − − − − 11_1666 lung 3 muAb 43-14A 0.2 μg/ml − − − − − − − 11_1666lung 3 muAb 43-14A 0.5 μg/ml − − − − − − − 11_1669 lung 4 muAb 43-14A0.2 μg/ml − − − − − − − 11_1669 lung 4 muAb 43-14A 0.5 μg/ml − − − − − −− 11_1672 lung 5 muAb 43-14A 0.2 μg/ml − − − − − − − 11_1672 lung 5 muAb43-14A 0.5 μg/ml − − − − − − − 11_1675 bronchial muAb 43-14A 0.2 μg/ml −− − − − − − 11_1675 bronchial muAb 43-14A 0.5 μg/ml − − − − − − −11_1678 bronchial muAb 43-14A 0.2 μg/ml − − − − − − − 11_1678 bronchialmuAb 43-14A 0.5 μg/ml − − − − − − − 11_1681 bronchial muAb 43-14A 0.2μg/ml − − − − − − − 11_1681 bronchial muAb 43-14A 0.5 μg/ml − − − − − −− 11_1684 stomach 9 muAb 43-14A 0.2 μg/ml +++ m >90 − n.a. − −

TABLE 8 Analysis of CLDN18.2 expression in esophageal, pancreatic andstomach cancerous tissues, using the monoclonal murine antibodies 43-14Aand 35-22A Details Tumor % positive Tissue ID Ab Tissue Subtype cellscells H/2010/10869 IVG 43-14A pancreas CA Acinar cell carcinoma − 01125005.22 43-14A pancreas CA Neuroendocrine ++ 70 carcinomaH/2006/22797** IA 43-14A pancreas CA Neuroendocrine − 0 carcinomaB08/6284-VC 35-22A pancreas CA Neuroendocrine − 0 carcinoma, NET G1B08/8549-4 35-22A pancreas CA Neuroendocrine − 0 carcinoma, NET G1B05/8523-3 35-22A pancreas CA PDAC ++ 1 B06/16136-NP2 35-22A pancreas CAPDAC ++ 50 B07/14168 35-22A pancreas CA PDAC ++ 80 B07/14935 35-22Apancreas CA PDAC − 0 B07/2633-III3 35-22A pancreas CA PDAC +++ 90B07/7430 35-22A pancreas CA PDAC ++ 90 B08/5618-2 35-22A pancreas CAPDAC +++ 80 B10/14198-VC 35-22A pancreas CA PDAC +++ 80 B10/706-VC335-22A pancreas CA PDAC +++ 60 B11/2059-D 35-22A pancreas CA PDAC +++ 80B11/4084 35-22A pancreas CA PDAC +++ 90 1125005.30 43-14A pancreas CAPDAC − 0 1125005.27 43-14A pancreas CA PDAC ++ 10 1125005.24 43-14Apancreas CA PDAC ++ 50 1125005.23 43-14A pancreas CA PDAC ++ 1001125005.25 43-14A pancreas CA PDAC +++ 30 1125005.28 43-14A pancreas CAPDAC +++ 80 H/2008/13074 43-14A pancreas CA PDAC +++ 40 H/2008/1319443-14A pancreas CA PDAC +++ 50 H/2008/380 43-14A pancreas CA PDAC +++ 90H/2009/11847 43-14A pancreas CA PDAC +++ 15 H/2009/20336 43-14A pancreasCA PDAC +++ 90 H/2009/23598 VII B 43-14A pancreas CA PDAC +++ 40H/2010/11569 43-14A pancreas CA PDAC +++ 80 H/2011/17191 VA 43-14Apancreas CA PDAC +++ 60 H/2009/4917 43-14A pancreas CA PDAC +++ 70H/2010/15941 43-14A pancreas CA PDAC +++ 50 H/2010/6709 43-14A pancreasCA PDAC +++ 70 1125005.19 43-14A esophagus CA adenocarcinoma ++ 50B09/1491-III-2 43-14A esophagus CA adenocarcinoma − 0 06/14957-2 43-14Aesophagus CA adenocarcinoma ++ 30 1125005.17 43-14A esophagus CAadenocarcinoma +++ 70 1083435B 43-14A esophagus CA adenocarcinoma ++ 7010b06684-II-3 43-14A stomach CA adenocarcinoma +++ 80 1125005.10 43-14Astomach CA adenocarcinoma ++ 30 1125005.6 43-14A stomach CAadenocarcinoma ++ 90 PDAC = pancreatic ductal adenocarcinoma

TABLE 9 Analysis of CLDN18.2 expression in lung cancerous tissues, usingmonoclonal murine antibody 43-14A Tumor cells B09/14758-3 0.2 μg/mlbronchiolo-alveolar type − — B09/18323-IV5 0.2 μg/ml bronchiolo-alveolartype − — B10/13211-VC4 0.2 μg/ml bronchiolo-alveolar type +++ 80%B07/4771-3 0.2 μg/ml carcinoid − — B07/5358 II2 0.2 μg/ml carcinoid − —B08/3382-1 0.2 μg/ml carcinoid − — B08/8898-II6 0.2 μg/ml carcinoma,clear cell + <1% B09/12293 II2 0.2 μg/ml carcinoma, spino cellular − —B06/12562-III3 0.2 μg/ml Carcinoma, large cell − — B06/10820-II2 0.2μg/ml carcinoma, adeno − — B06/10876-2 0.2 μg/ml carcinoma, adeno − —B06/16831-I3 0.2 μg/ml carcinoma, adeno +++ 80% B07/03255 IV5 0.2 μg/mlcarcinoma, adeno +++  5% B07/2296-3 0.2 μg/ml carcinoma, adeno − —B07/709 II4 0.2 μg/ml carcinoma, adeno ++ <5% B10/12713-II1 0.2 μg/mlcarcinoma, adeno ++ <5% B10/14197-2 0.2 μg/ml carcinoma, adeno − —B10/16367-V2 0.2 μg/ml carcinoma, adeno +  1% B09/1743 II3 0.2 μg/mlcarcinoma, adeno, − — squamous B09/17916-1 0.2 μg/ml carcinoma, largecell − — B10/10814-3 0.2 μg/ml carcinoma, large cell +  1% neuroendocrine B010/10646 0.2 μg/ml carcinoma, squamous cell − — B06/3204-20.2 μg/ml carcinoma, squamous cell − — B08/292-2 0.2 μg/ml carcinoma,squamous cell ++ <5% B08/7434-IV 0.2 μg/ml carcinoma, squamous cell − —B09/45-2 0.2 μg/ml carcinoma, squamous cell − — B10/10-2 0.2 μg/mlcarcinoma, squamous cell + <1% B10/11714 0.2 μg/ml carcinoma, squamouscell − — B10/15779 0.2 μg/ml carcinoma, squamous cell − — B10/17043 0.2μg/ml carcinoma, squamous cell − — B10/18106 0.2 μg/ml carcinoma,squamous cell − — B07/6782-1 0.2 μg/ml carcinoma; adeno, ++ <1% clearcell B07/9741-5 0.2 μg/ml carcinoma; adeno, − — clear cell B08/16425-20.2 μg/ml large cell neuro-endocrine ++ <5% B08/3019-V3 0.2 μg/mlnon-small cell CA − — B08/1099 V3 0.2 μg/ml nt small cell long CA − <1%B08/12010-2 0.2 μg/ml nt small cell long CA ++ <1% B10/17662-III3 0.2μg/ml small cell carcinoma − —

The invention claimed is:
 1. An antibody or antigen binding fragmentthereof that binds to claudin 18.2 (CLDN18.2) or a peptide having theamino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQID NO: 6) wherein the antibody or antigen-binding fragment comprises (i)an antibody heavy chain comprising: a CDR1 having the amino acidsequence set forth in SEQ ID NO: 8, a CDR2 having the amino acidsequence set forth in SEQ ID NO: 9, and a CDR3 having the amino acidsequence set forth in SEQ ID NO: 10; and an antibody light chaincomprising: a CDR1 having the amino acid sequence set forth in SEQ IDNO: 12, a CDR2 having the amino acid sequence set forth in SEQ ID NO:13, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:14; or (ii) an antibody heavy chain comprising: a CDR1 having the aminoacid sequence set forth in SEQ ID NO: 16, a CDR2 having the amino acidsequence set forth in SEQ ID NO: 17, and a CDR3 having the amino acidsequence set forth in SEQ ID NO: 18; and an antibody light chaincomprising: a CDR1 having the amino acid sequence set forth in SEQ IDNO: 20, a CDR2 having the amino acid sequence set forth in SEQ ID NO:21, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:22.
 2. The antibody or antigen-binding fragment thereof of claim 1,wherein said CLDN18.2 is cell surface membrane-bound CLDN18.2.
 3. Theantibody or antigen-binding fragment thereof of claim 1, wherein saidCLDN18.2 is present on cancer cells.
 4. The antibody or antigen-bindingfragment thereof of claim 3, wherein said cancer cells are CLDN18.2expressing cancer cells.
 5. The antibody or antigen-binding fragmentthereof of claim 3, wherein said cancer cells are selected from thegroup consisting of gastric, esophageal, pancreatic, lung, ovarian,colon, hepatic, head-neck, and gallbladder cancer cells.
 6. The antibodyor antigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof does not bind to non-cancerous cellsexcept stomach epithelial cells.
 7. The antibody or antigen-bindingfragment thereof of claim 1, wherein the antibody or antigen-bindingfragment thereof does not bind to non-cancerous lung cells.
 8. Theantibody of claim 1, wherein the antibody is a chimeric, human orhumanized antibody.
 9. The antibody of claim 1, wherein the antibody isa monoclonal antibody.
 10. A conjugate comprising an antibody orantigen-binding fragment of claim 1 coupled to at least one detectablelabel.
 11. A hybridoma capable of producing the antibody of claim
 1. 12.The antibody or antigen-binding fragment of claim 1, wherein saidCLDN18.2 comprises the amino acid sequence according to SEQ ID NO: 2 ofthe sequence listing or a variant of said amino acid sequence.
 13. Theantibody or antigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment comprises an antibody heavy chain comprising: aCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, a CDR2having the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3having the amino acid sequence set forth in SEQ ID NO: 18; and anantibody light chain comprising: a CDR1 having the amino acid sequenceset forth in SEQ ID NO: 20, a CDR2 having the amino acid sequence setforth in SEQ ID NO: 21, and a CDR3 having the amino acid sequence setforth in SEQ ID NO:
 22. 14. The antibody or antigen-binding fragment ofclaim 1, wherein the antibody or antigen-binding fragment comprises anantibody heavy chain comprising: a CDR1 having the amino acid sequenceset forth in SEQ ID NO: 8, a CDR2 having the amino acid sequence setforth in SEQ ID NO: 9, and a CDR3 having the amino acid sequence setforth in SEQ ID NO: 10; and an antibody light chain comprising: a CDR1having the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 havingthe amino acid sequence set forth in SEQ ID NO: 13, and a CDR3 havingthe amino acid sequence set forth in SEQ ID NO:
 14. 15. The antibody orantigen-binding fragment of claim 13, wherein the antibody orantigen-binding fragment comprises an antibody heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO: 15, and an antibodylight chain comprising the amino acid sequence set forth in SEQ ID NO:19.
 16. The antibody or antigen-binding fragment of claim 14, whereinthe antibody or antigen-binding fragment comprises an antibody heavychain comprising the amino acid sequence set forth in SEQ ID NO: 7, andan antibody light chain comprising the amino acid sequence set forth inSEQ ID NO:
 11. 17. A diagnostic test kit which comprises the antibody orantigen-binding fragment of claim
 1. 18. A method for detecting CLDN18.2or determining the quantity of CLDN18.2 in a sample comprising the stepsof: (i) contacting a sample with the antibody or antigen-bindingfragment of claim 1 and (ii) detecting the formation of a complex ordetermining the quantity of a complex between the antibody or theantigen-binding fragment and CLDN18.2.
 19. A method for determiningwhether cells express CLDN18.2 comprising the steps of: (i) contacting acellular sample with the antibody or antigen-binding fragment of claim 1and (ii) detecting the formation of a complex between the antibody orthe antigen-binding fragment and CLDN18.2 expressed by cells in saidsample.
 20. A method for diagnosis, detection or monitoring of cancercomprising the steps of: contacting a biological sample with theantibody or antigen-binding fragment of claim 1 and (ii) detecting theformation of a complex and/or determining the quantity of a complexbetween the antibody or the antigen-binding fragment and CLDN18.2.
 21. Amethod for determining whether a cancer is treatable by a cancer therapytargeting CLDN18.2 comprising the steps of: (i) contacting a samplecomprising cancer cells with the antibody or antigen-binding fragment ofclaim 1 and (ii) detecting the formation of a complex between theantibody or the antigen-binding fragment and CLDN18.2.
 22. The method ofclaim 18, wherein the sample comprises a paraffin embedded tissuesection fixed with paraformaldehyde.
 23. The method of claim 18,wherein, in step (i), the antibody or antigen binding fragment is at aconcentration of about 0.1 to about 0.5 μg/ml.
 24. The method of claim23, wherein the concentration is about 0.2 μg/ml.
 25. The method ofclaim 18, wherein step (i) comprises incubating the sample with theantibody or antigen-binding fragment overnight at 4° C.
 26. The methodof claim 18, wherein the antibody or antigen binding fragment is labeledwith a detectable label.
 27. The method of claim 26, wherein thedetectable label is an enzyme or a fluorophore.
 28. The method of claim18, wherein the method further comprises contacting the antibody orantigen-binding fragment with a secondary antibody, wherein thesecondary antibody is labeled with a detectable label.
 29. The method ofclaim 28, wherein the secondary antibody is a horseradish-peroxidase(HRP)-labeled secondary antibody.
 30. The method of claim 29, whereinstep (ii) comprises exposing the HRP-labeled secondary antibody to achemiluminescent substrate.
 31. The method of claim 18, wherein theantibody or antigen-binding fragment comprises an antibody heavy chaincomprising: a CDR1 having the amino acid sequence set forth in SEQ IDNO: 8, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 9,and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 10;and an antibody light chain comprising: a CDR1 having the amino acidsequence set forth in SEQ ID NO: 12, a CDR2 having the amino acidsequence set forth in SEQ ID NO: 13, and a CDR3 having the amino acidsequence set forth in SEQ ID NO:
 14. 32. The method of claim 18, whereinthe antibody or antigen-binding fragment comprises an antibody heavychain comprising the amino acid sequence set forth in SEQ ID NO: 7, andan antibody light chain comprising the amino acid sequence set forth inSEQ ID NO:
 11. 33. The method of claim 18, wherein the antibody orantigen-binding fragment comprises an antibody heavy chain comprising: aCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, a CDR2having the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3having the amino acid sequence set forth in SEQ ID NO: 18; and anantibody light chain comprising: a CDR1 having the amino acid sequenceset forth in SEQ ID NO: 20, a CDR2 having the amino acid sequence setforth in SEQ ID NO: 21, and a CDR3 having the amino acid sequence setforth in SEQ ID NO:
 22. 34. The method of claim 18, wherein the antibodyor antigen-binding fragment comprises an antibody heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO: 15, and an antibodylight chain comprising the amino acid sequence set forth in SEQ ID NO:19.